1 5706 122 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 2 22 46 5-HYDROXYMETHYLCYTOSINE AS A CLINICAL BIOMARKER: FLUORESCENCE-BASED ASSAY FOR HIGH-THROUGHPUT EPIGENETIC QUANTIFICATION IN HUMAN TISSUES. EPIGENETIC TRANSFORMATIONS MAY PROVIDE EARLY INDICATORS FOR CANCER AND OTHER DISEASE. SPECIFICALLY, THE AMOUNT OF GENOMIC 5-HYDROXYMETHYLCYTOSINE (5-HMC) WAS SHOWN TO BE GLOBALLY REDUCED IN A WIDE RANGE OF CANCERS. THE INTEGRATION OF THIS GLOBAL BIOMARKER INTO DIAGNOSTIC WORKFLOWS IS HAMPERED BY THE LIMITATIONS OF CURRENT 5-HMC QUANTIFICATION METHODS. HERE WE PRESENT AND VALIDATE A FLUORESCENCE-BASED PLATFORM FOR HIGH-THROUGHPUT AND COST-EFFECTIVE QUANTIFICATION OF GLOBAL GENOMIC 5-HMC LEVELS. WE UTILIZED THE ASSAY TO CHARACTERIZE CANCEROUS TISSUES BASED ON THEIR 5-HMC CONTENT, AND OBSERVED A PRONOUNCED REDUCTION IN 5-HMC LEVEL IN VARIOUS CANCER TYPES. WE PRESENT DATA FOR GLIOBLASTOMA, COLORECTAL CANCER, MULTIPLE MYELOMA, CHRONIC LYMPHOCYTIC LEUKEMIA AND PANCREATIC CANCER, COMPARED TO CORRESPONDING CONTROLS. POTENTIALLY, THE TECHNIQUE COULD ALSO BE USED TO FOLLOW RESPONSE TO TREATMENT FOR PERSONALIZED TREATMENT SELECTION. WE PRESENT INITIAL PROOF-OF-CONCEPT DATA FOR TREATMENT OF FAMILIAL ADENOMATOUS POLYPOSIS. 2020 3 2920 44 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 4 1794 35 EFFECT OF DIABETES STATUS AND HYPERGLYCEMIA ON GLOBAL DNA METHYLATION AND HYDROXYMETHYLATION. TYPE 2 DIABETES MELLITUS (T2DM) IS CHARACTERIZED BY OXIDATIVE STRESS THAT COULD LEAD TO CHRONIC MICRO- AND MACROVASCULAR COMPLICATIONS. WE HYPOTHESIZED THAT SOME OF THE TARGET ORGAN DAMAGE IS MEDIATED BY OXIDATIVE ALTERATIONS IN EPIGENETIC MECHANISMS INVOLVING DNA METHYLATION (5MC) AND DNA HYDROXYMETHYLATION (5HMC). WE ANALYZED GLOBAL DNA METHYLATION AND HYDROXYMETHYLATION IN PERIPHERAL BLOOD CELLS IN WELL-CONTROLLED AND POORLY CONTROLLED PATIENTS WITH T2DM AND COMPARED THEM WITH HEALTHY CONTROLS. WE ALSO ANALYZED MICROARRAYS OF DNA METHYLATION AND GENE EXPRESSION OF OTHER IMPORTANT TISSUES IN THE CONTEXT OF DIABETES FROM THE GEO DATABASE REPOSITORY AND THEN COMPARED THESE RESULTS WITH OUR EXPERIMENTAL GENE EXPRESSION DATA. DNA METHYLATION AND, MORE IMPORTANTLY, DNA HYDROXYMETHYLATION LEVELS WERE INCREASED IN POORLY CONTROLLED PATIENTS COMPARED TO WELL-CONTROLLED AND HEALTHY INDIVIDUALS. BOTH 5MC AND 5HMC MEASUREMENTS WERE CORRELATED WITH THE PERCENTAGE OF GLYCATED HEMOGLOBIN, INDICATING A DIRECT IMPACT OF HYPERGLYCEMIA ON CHANGES OVER THE EPIGENOME. THE ANALYSIS OF METHYLATION MICROARRAYS WAS CONCORDANT, AND 5MC LEVELS WERE INCREASED IN THE PERIPHERAL BLOOD OF T2DM PATIENTS. HOWEVER, THE DNA METHYLATION LEVELS WERE THE OPPOSITE OF THOSE IN OTHER TISSUES, SUCH AS THE PANCREAS, ADIPOSE TISSUE AND SKELETAL MUSCLE. WE HYPOTHESIZE THAT A PROCESS OF DNA OXIDATION ASSOCIATED WITH HYPERGLYCEMIA MAY EXPLAIN THE DNA DEMETHYLATION IN WHICH THE ACTIVITY OF TEN-ELEVEN TRANSLOCATION (TET) PROTEINS IS NOT SUFFICIENT TO COMPLETE THE PROCESS. HIGH LEVELS OF GLUCOSE LEAD TO CELLULAR OXIDATION, WHICH TRIGGERS THE PROCESS OF DNA DEMETHYLATION AIDED BY TET ENZYMES, RESULTING IN EPIGENETIC DYSREGULATION OF THE DAMAGED TISSUES. 2017 5 70 28 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 6 1577 29 DNA METHYLATION PROFILE IN CHRONIC MYELOMONOCYTIC LEUKEMIA ASSOCIATES WITH DISTINCT CLINICAL, BIOLOGICAL AND GENETIC FEATURES. CHROMOSOMAL ABNORMALITIES ARE DETECTED IN 20-30% OF PATIENTS WITH CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML) AND CORRELATE WITH PROGNOSIS. ON THE MUTATION LEVEL, DISRUPTIVE ALTERATIONS ARE PARTICULARLY FREQUENT IN CHROMATIN REGULATORY GENES. HOWEVER, LITTLE IS KNOWN ABOUT THE CONSEQUENTIAL ALTERATIONS IN THE EPIGENETIC MARKING OF THE GENOME. HERE, WE REPORT THE ANALYSIS OF GENOMIC DNA METHYLATION PATTERNS OF 64 CMML PATIENTS AND 10 HEALTHY CONTROLS, USING A DNA METHYLATION MICROARRAY FOCUSED ON PROMOTER REGIONS. DIFFERENTIAL METHYLATION ANALYSIS BETWEEN PATIENTS AND CONTROLS ALLOWED US TO IDENTIFY ABNORMALITIES IN DNA METHYLATION, INCLUDING HYPERMETHYLATION OF SPECIFIC GENES AND LARGE GENOME REGIONS WITH ABERRANT DNA METHYLATION. UNSUPERVISED HIERARCHICAL CLUSTER ANALYSIS IDENTIFIED TWO MAIN CLUSTERS THAT ASSOCIATED WITH THE CLINICAL, BIOLOGICAL, AND GENETIC FEATURES OF PATIENTS. GROUP 1 WAS ENRICHED IN PATIENTS WITH ADVERSE CLINICAL AND BIOLOGICAL CHARACTERISTICS AND POORER OVERALL AND PROGRESSION-FREE SURVIVAL. IN ADDITION, SIGNIFICANT DIFFERENCES IN DNA METHYLATION WERE OBSERVED BETWEEN PATIENTS WITH LOW RISK AND INTERMEDIATE/HIGH RISK KARYOTYPES AND BETWEEN TET2 MUTANT AND WILD TYPE PATIENTS. TAKEN TOGETHER, OUR RESULTS DEMONSTRATE THAT ALTERED DNA METHYLATION PATTERNS REFLECT THE CMML DISEASE STATE AND ALLOW TO IDENTIFY PATIENT GROUPS WITH DISTINCT CLINICAL FEATURES. 2018 7 287 27 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 8 839 44 CHEMO-ENZYMATIC FLUORESCENCE LABELING OF GENOMIC DNA FOR SIMULTANEOUS DETECTION OF GLOBAL 5-METHYLCYTOSINE AND 5-HYDROXYMETHYLCYTOSINE. 5-METHYLCYTOSINE AND 5-HYDROXYMETHYLCYTOSINE ARE EPIGENETIC MODIFICATIONS INVOLVED IN GENE REGULATION AND CANCER. WE PRESENT A NEW, SIMPLE, AND HIGH-THROUGHPUT PLATFORM FOR MULTI-COLOR EPIGENETIC ANALYSIS. THE NOVELTY OF OUR APPROACH IS THE ABILITY TO MULTIPLEX METHYLATION AND DE-METHYLATION SIGNALS IN THE SAME ASSAY. WE UTILIZE AN ENGINEERED METHYLTRANSFERASE ENZYME THAT RECOGNIZES AND LABELS ALL UNMODIFIED CPG SITES WITH A FLUORESCENT COFACTOR. IN COMBINATION WITH THE ALREADY ESTABLISHED LABELING OF THE DE-METHYLATION MARK 5-HYDROXYMETHYLCYTOSINE VIA ENZYMATIC GLYCOSYLATION, WE OBTAINED A ROBUST PLATFORM FOR SIMULTANEOUS EPIGENETIC ANALYSIS OF THESE MARKS. WE ASSESSED THE GLOBAL EPIGENETIC LEVELS IN MULTIPLE SAMPLES OF COLORECTAL CANCER AND OBSERVED A 3.5-FOLD REDUCTION IN 5HMC LEVELS BUT NO CHANGE IN DNA METHYLATION LEVELS BETWEEN SICK AND HEALTHY INDIVIDUALS. WE ALSO MEASURED EPIGENETIC MODIFICATIONS IN CHRONIC LYMPHOCYTIC LEUKEMIA AND OBSERVED A DECREASE IN BOTH MODIFICATION LEVELS (5-HYDROXYMETHYLCYTOSINE: WHOLE BLOOD 30 %; PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMCS) 40 %. 5-METHYLCYTOSINE: WHOLE BLOOD 53 %; PBMCS 48 %). OUR FINDINGS PROPOSE USING A SIMPLE BLOOD TEST AS A VIABLE METHOD FOR ANALYSIS, SIMPLIFYING SAMPLE HANDLING IN DIAGNOSTICS. IMPORTANTLY, OUR RESULTS HIGHLIGHT THE ASSAY'S POTENTIAL FOR EPIGENETIC EVALUATION OF CLINICAL SAMPLES, BENEFITING RESEARCH AND PATIENT MANAGEMENT. 2023 9 1583 31 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 10 3686 22 INFLAMMATION-RELATED ABERRANT PATTERNS OF DNA METHYLATION: DETECTION AND ROLE IN EPIGENETIC DEREGULATION OF CANCER CELL TRANSCRIPTOME. IT IS NOW APPARENT THAT EPIGENETIC ABNORMALITIES, IN PARTICULAR ALTERED DNA METHYLATION, PLAY A CRUCIAL ROLE IN THE DEVELOPMENT AND PROGRESSION OF HUMAN CANCERS. DNA HYPERMETHYLATION AT PROMOTER CPG ISLANDS IS NOW RECOGNIZED AS A THIRD MECHANISM BY WHICH INACTIVATION OF TUMOR SUPPRESSOR GENES OCCURS. ABERRANT CPG ISLAND HYPERMETHYLATION IS ALSO FREQUENTLY OBSERVED IN CHRONIC INFLAMMATION AND PRECANCEROUS LESIONS, WHICH SUGGESTS THAT IT IS AN EARLY EVENT IN TUMORIGENESIS THAT COULD SERVE AS A USEFUL TUMOR MARKER. A VARIETY OF SCREENING TECHNIQUES HAVE BEEN DEVELOPED FOR GENOME-WIDE SCREENING OF METHYLATION STATUS. OF THOSE, TRANSCRIPTOME ANALYSIS COUPLED WITH PHARMACOLOGICAL UNMASKING HAS EMERGED AS A POWERFUL TOOL FOR REVEALING DNA METHYLATION PATTERNS IN CANCER CELLS AND IDENTIFYING NEW TUMOR MARKER CANDIDATES. 2009 11 3503 29 IDENTIFICATION OF POTENTIAL DIFFERENTIALLY METHYLATED GENE-RELATED BIOMARKERS IN ENDOMETRIOSIS. AIM: TO IDENTIFY EPIGENETIC ALTERATIONS OF DIFFERENTIALLY EXPRESSED GENES AND SCREEN OUT TARGETED THERAPEUTIC DRUGS IN ENDOMETRIOSIS. METHODS: BASED ON THE GENE EXPRESSION OMNIBUS DATABASE AND A SERIES OF BIOLOGICAL INFORMATION ANALYSIS TOOLS, SUPPLEMENTED BY VALIDATION OF CLINICAL SAMPLES, ABERRANT DNA METHYLATION-DRIVEN GENES AND THEIR FUNCTIONS WERE EXPLORED, AS WELL AS POSSIBLE TARGETED DRUGS. RESULTS: THIS STUDY SCREENED OUT A RANGE OF DNA METHYLATION-DRIVEN GENES THAT WERE ASSOCIATED WITH POWERFUL PROPERTIES AND CORRESPONDING PATHWAYS. AMONG THEM, BDNF AND CCL2 WERE KEY GENES IN THE DEVELOPMENT OF ENDOMETRIOSIS. FOUR CHEMICAL AGENTS HAVE BEEN FLAGGED AS POTENTIAL TREATMENTS FOR ENDOMETRIOSIS. CONCLUSION: THESE CANDIDATE GENES AND SMALL-MOLECULE AGENTS MAY BE FURTHER EXPLORED AS POTENTIAL TARGETS AND DRUGS FOR ENDOMETRIOSIS DIAGNOSIS AND THERAPY, RESPECTIVELY. 2022 12 2771 32 EXTENSIVE PROMOTER DNA HYPERMETHYLATION AND HYPOMETHYLATION IS ASSOCIATED WITH ABERRANT MICRORNA EXPRESSION IN CHRONIC LYMPHOCYTIC LEUKEMIA. DYSREGULATED MICRORNA (MIRNA) EXPRESSION CONTRIBUTES TO THE PATHOGENESIS OF HEMATOPOIETIC MALIGNANCIES, INCLUDING CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). HOWEVER, AN UNDERSTANDING OF THE MECHANISMS THAT CAUSE ABERRANT MIRNA TRANSCRIPTIONAL CONTROL IS LACKING. IN THIS STUDY, WE COMPREHENSIVELY INVESTIGATED THE ROLE AND EXTENT OF MIRNA EPIGENETIC REGULATION IN CLL. GENOME-WIDE PROFILING CONDUCTED ON 24 CLL AND 10 HEALTHY B CELL SAMPLES REVEALED GLOBAL DNA METHYLATION PATTERNS UPSTREAM OF MIRNA SEQUENCES THAT DISTINGUISHED MALIGNANT FROM HEALTHY CELLS AND IDENTIFIED PUTATIVE MIRNA PROMOTERS. INTEGRATION OF DNA METHYLATION AND MIRNA PROMOTER DATA LED TO THE IDENTIFICATION OF 128 RECURRENT MIRNA TARGETS FOR ABERRANT PROMOTER DNA METHYLATION. DNA HYPOMETHYLATION ACCOUNTED FOR MORE THAN 60% OF ALL ABERRANT PROMOTER-ASSOCIATED DNA METHYLATION IN CLL, AND PROMOTER DNA HYPOMETHYLATION WAS RESTRICTED TO WELL-DEFINED REGIONS. INDIVIDUAL HYPER- AND HYPOMETHYLATED PROMOTERS ALLOWED DISCRIMINATION OF CLL SAMPLES FROM HEALTHY CONTROLS. PROMOTER DNA METHYLATION PATTERNS WERE CONFIRMED IN AN INDEPENDENT PATIENT COHORT, WITH 11 MIRNAS CONSISTENTLY SHOWING AN INVERSE CORRELATION BETWEEN DNA METHYLATION STATUS AND EXPRESSION LEVEL. TOGETHER, OUR FINDINGS CHARACTERIZE THE ROLE OF EPIGENETIC CHANGES IN THE REGULATION OF MIRNA TRANSCRIPTION AND CREATE A REPOSITORY OF DISEASE-SPECIFIC PROMOTER REGIONS THAT MAY PROVIDE ADDITIONAL INSIGHTS INTO THE PATHOGENESIS OF CLL. 2012 13 1582 32 DNA METHYLATION PROFILES IN PRECANCEROUS TISSUE AND CANCERS: CARCINOGENETIC RISK ESTIMATION AND PROGNOSTICATION BASED ON DNA METHYLATION STATUS. ALTERATIONS IN DNA METHYLATION, WHICH ARE ASSOCIATED WITH DNA METHYLTRANSFERASE ABNORMALITIES AND RESULT IN SILENCING OF TUMOR-RELATED GENES AND CHROMOSOMAL INSTABILITY, ARE INVOLVED EVEN IN PRECANCEROUS CHANGES IN VARIOUS ORGANS. DNA METHYLATION ALTERATIONS ALSO ACCOUNT FOR THE HISTOLOGICAL HETEROGENEITY AND CLINICOPATHOLOGICAL DIVERSITY OF HUMAN CANCERS. THEREFORE, WE HAVE ANALYZED DNA METHYLATION ON A GENOME-WIDE SCALE IN CLINICAL TISSUE SAMPLES. OUR APPROACH USING THE BACTERIAL ARTIFICIAL CHROMOSOME ARRAY-BASED METHYLATED CPG ISLAND AMPLIFICATION METHOD HAS REVEALED THAT DNA METHYLATION ALTERATIONS CORRELATED WITH THE FUTURE DEVELOPMENT OF MORE MALIGNANT CANCERS ARE ALREADY ACCUMULATED AT THE PRECANCEROUS STAGE IN THE KIDNEY, LIVER AND URINARY TRACT. DNA METHYLATION PROFILES AT PRECANCEROUS STAGES ARE BASICALLY INHERITED BY THE CORRESPONDING CANCERS DEVELOPING IN INDIVIDUAL PATIENTS. SUCH DNA METHYLATION ALTERATIONS MAY CONFER VULNERABILITY TO FURTHER GENETIC AND EPIGENETIC ALTERATIONS, GENERATE MORE MALIGNANT CANCERS, AND THUS DETERMINE PATIENT OUTCOME. ON THE BASIS OF BACTERIAL ARTIFICIAL CHROMOSOME ARRAY-BASED METHYLATED CPG ISLAND AMPLIFICATION DATA, INDICATORS FOR CARCINOGENETIC RISK ESTIMATION HAVE BEEN ESTABLISHED USING LIVER TISSUE SPECIMENS FROM PATIENTS WITH HEPATITIS VIRUS INFECTION, CHRONIC HEPATITIS AND LIVER CIRRHOSIS OR HISTOLOGICALLY NORMAL UROTHELIA, AND FOR PROGNOSTICATION USING BIOPSY OR SURGICALLY RESECTED SPECIMENS FROM PATIENTS WITH RENAL CELL CARCINOMA, HEPATOCELLULAR CARCINOMA AND UROTHELIAL CARCINOMA. SUCH GENOME-WIDE DNA METHYLATION PROFILING HAS NOW FIRMLY ESTABLISHED THE CLINICAL RELEVANCE OF TRANSLATIONAL EPIGENETICS. 2010 14 3067 27 GENOME-WIDE DNA METHYLATION PROFILES IN PRECANCEROUS CONDITIONS AND CANCERS. ALTERATIONS OF DNA METHYLATION, WHICH RESULT IN CHROMOSOMAL INSTABILITY AND SILENCING OF TUMOR-RELATED GENES, ARE AMONG THE MOST CONSISTENT EPIGENETIC CHANGES OBSERVED IN HUMAN CANCERS. ANALYSIS OF TISSUE SPECIMENS HAS REVEALED THAT DNA METHYLATION ALTERATIONS PARTICIPATE IN MULTISTAGE CARCINOGENESIS, EVEN FROM THE EARLY AND PRECANCEROUS STAGES, ESPECIALLY IN ASSOCIATION WITH CHRONIC INFLAMMATION AND/OR PERSISTENT VIRAL INFECTION, SUCH AS CHRONIC HEPATITIS OR LIVER CIRRHOSIS RESULTING FROM INFECTION WITH HEPATITIS B OR C VIRUS. DNA METHYLATION ALTERATIONS CAN ACCOUNT FOR THE HISTOLOGICAL HETEROGENEITY AND CLINICOPATHOLOGICAL DIVERSITY OF HUMAN CANCERS. OVEREXPRESSION OF DNA METHYLTRANSFERASE 1 IS NOT A SECONDARY RESULT OF INCREASED CELL PROLIFERATIVE ACTIVITY, BUT IS SIGNIFICANTLY CORRELATED WITH ACCUMULATION OF DNA HYPERMETHYLATION IN CPG ISLANDS OF TUMOR-RELATED GENES. ALTERATION OF DNA METHYLTRANSFERASE 3B SPLICING MAY RESULT IN CHROMOSOMAL INSTABILITY THROUGH DNA HYPOMETHYLATION IN PERICENTROMERIC SATELLITE REGIONS. GENOME-WIDE ANALYSIS OF DNA METHYLATION STATUS HAS REVEALED THAT THE DNA METHYLATION PROFILE AT THE PRECANCEROUS STAGE IS BASICALLY INHERITED BY THE CORRESPONDING CANCERS DEVELOPING IN INDIVIDUAL PATIENTS. DNA METHYLATION STATUS IS NOT SIMPLY ALTERED AT THE PRECANCEROUS STAGE; RATHER, DNA METHYLATION ALTERATIONS AT THE PRECANCEROUS STAGE MAY CONFER VULNERABILITY TO FURTHER GENETIC AND EPIGENETIC ALTERATIONS, GENERATE MORE MALIGNANT CANCERS, AND THUS DETERMINE PATIENT OUTCOME. THEREFORE, GENOME-WIDE DNA METHYLATION PROFILING MAY PROVIDE OPTIMAL INDICATORS FOR CARCINOGENETIC RISK ESTIMATION AND PROGNOSTICATION, AND THUS PROVIDE AN AVENUE FOR CANCER PREVENTION AND THERAPY ON AN INDIVIDUAL BASIS. 2010 15 1307 25 DEFINING A METHYLATION SIGNATURE ASSOCIATED WITH OPERATIONAL TOLERANCE IN KIDNEY TRANSPLANT RECIPIENTS. OPERATIONAL TOLERANCE AFTER KIDNEY TRANSPLANTATION IS DEFINED AS STABLE GRAFT ACCEPTANCE WITHOUT THE NEED FOR IMMUNOSUPPRESSION THERAPY. HOWEVER, IT IS NOT CLEAR WHICH CELLULAR AND MOLECULAR PATHWAYS ARE DRIVING TOLERANCE IN THESE PATIENTS. WE PERFORMED GENOME-WIDE ANALYSIS OF DNA METHYLATION IN PERIPHERAL BLOOD MONONUCLEAR CELLS FROM KIDNEY TRANSPLANT RECIPIENTS WITH CHRONIC REJECTION AND OPERATIONAL TOLERANCE FROM THE GENETIC ANALYSIS OF MOLECULAR BIOMARKERS OF IMMUNOLOGICAL TOLERANCE (GAMBIT) STUDY. OUR RESULTS SHOWED THAT BOTH CLINICAL STAGES DIVERGE IN 2737 GENES, INDICATING THAT EACH ONE HAS A SPECIFIC METHYLATION SIGNATURE ASSOCIATED WITH TRANSPLANT OUTCOME. WE ALSO OBSERVED THAT TOLERANCE IS ASSOCIATED WITH DEMETHYLATION IN GENES INVOLVED IN IMMUNE FUNCTION, INCLUDING B AND T CELL ACTIVATION AND TH17 DIFFERENTIATION, WHILE IN CHRONIC REJECTION IT IS ASSOCIATED WITH INTRACELLULAR SIGNALING AND UBIQUITINATION PATHWAYS. USING CO-EXPRESSION NETWORK ANALYSIS, WE SELECTED 12 GENOMIC REGIONS THAT ARE SPECIFICALLY HYPOMETHYLATED OR HYPERMETHYLATED IN TOLERANT PATIENTS. ANALYSIS OF THESE GENES IN TRANSPLANTED PATIENTS WITH LOW DOSE OF STEROIDS SHOWED THAT THESE HAVE A SIMILAR METHYLATION SIGNATURE TO THAT OF TOLERANT RECIPIENTS. OVERALL, THESE RESULTS DEMONSTRATE THAT METHYLATION ANALYSIS CAN MIRROR THE IMMUNE STATUS ASSOCIATED WITH TRANSPLANT OUTCOME AND PROVIDES A STARTING POINT FOR UNDERSTANDING THE EPIGENETIC MECHANISMS ASSOCIATED WITH TOLERANCE. 2021 16 2682 34 EVALUATION OF SERUM LINE-1 HYPOMETHYLATION AS A PROGNOSTIC MARKER FOR HEPATOCELLULAR CARCINOMA. BACKGROUND AND STUDY AIMS: GLOBAL HYPOMETHYLATION IS ONE OF THE MOST CONSISTENT EPIGENETIC CHANGES IN CANCER. DEVELOPMENT OF HEPATOCELLULAR CARCINOMA (HCC) MUST BE UNDERSTOOD AS A MULTISTEP PROCESS WITH ACCUMULATION OF GENETIC AND EPIGENETIC ALTERATIONS. IN THE LAST DECADES, IN ADDITION TO GENETIC ALTERATIONS, EPIGENETIC CHANGES HAVE BEEN RECOGNIZED AS AN IMPORTANT AND ALTERNATIVE MECHANISM IN TUMOURIGENESIS. WE INVESTIGATED THE CLINICAL IMPLICATIONS OF GLOBAL HYPOMETHYLATION IN THE SERA OF PATIENTS WITH HEPATOCELLULAR CARCINOMA (HCC). PATIENTS AND METHODS: PCR WAS USED TO ASSESS THE METHYLATION STATUS OF LONG INTERSPERSED NUCLEAR ELEMENT TYPE 1 (LINE-1) REPETITIVE SEQUENCES IN GENOMIC DNA DERIVED FROM SERA OF 50 PATIENTS WITH HCC, 20 PATIENTS WITH CIRRHOSIS, 20 PATIENTS WITH CHRONIC HEPATITIS C AND 10 HEALTHY SUBJECTS. RESULTS: SERUM GENOME HYPOMETHYLATION WAS SIGNIFICANTLY INCREASED IN PATIENTS WITH HCC (P<0.001). THE LEVELS OF SERUM LINE-1 HYPOMETHYLATION AT INITIAL PRESENTATION CORRELATED SIGNIFICANTLY WITH TUMOUR SIZE, TUMOUR NUMBER AND ALPHA-FOETOPROTEIN LEVEL. MOREOVER HIGH SERUM LINE-1 HYPOMETHYLATION CORRELATES SIGNIFICANTLY WITH POOR SURVIVAL. CONCLUSION: SERUM LINE-1 HYPOMETHYLATION MAY SERVE AS A PROGNOSTIC MARKER FOR PATIENTS WITH HCC. 2011 17 3 36 "EPIGENOME-WIDE METHYLATION PROFILE OF CHRONIC KIDNEY DISEASE-DERIVED ARTERIAL DNA UNCOVERS NOVEL PATHWAYS IN DISEASE-ASSOCIATED CARDIOVASCULAR PATHOLOGY.". CHRONIC KIDNEY DISEASE (CKD) RELATED CARDIOVASCULAR DISEASE (CVD) IS CHARACTERIZED BY VASCULAR REMODELLING WITH WELL-ESTABLISHED STRUCTURAL AND FUNCTIONAL CHANGES IN THE VASCULAR WALL SUCH AS ARTERIAL STIFFNESS, MATRIX DEPOSITION, AND CALCIFICATION. THESE PHENOTYPIC CHANGES RESEMBLE PATHOLOGY SEEN IN AGEING, AND ARE LIKELY TO BE MEDIATED BY SUSTAINED ALTERATIONS IN GENE EXPRESSION, WHICH MAY BE CAUSED BY EPIGENETIC CHANGES SUCH AS TISSUE-SPECIFIC DNA METHYLATION. WE AIMED TO INVESTIGATE TISSUE SPECIFIC CHANGES IN DNA METHYLATION THAT OCCUR IN CKD-RELATED CVD. GENOME-WIDE DNA METHYLATION CHANGES WERE EXAMINED IN BISULPHITE CONVERTED GENOMIC DNA ISOLATED FROM THE VASCULAR MEDIA OF CKD AND HEALTHY ARTERIES. METHYLATION-SPECIFIC PCR WAS USED TO VALIDATE THE ARRAY DATA, AND THE ASSOCIATION BETWEEN DNA METHYLATION AND GENE AND PROTEIN EXPRESSION WAS EXAMINED. THE DNA METHYLATION AGE WAS COMPARED TO THE CHRONOLOGICAL AGE IN BOTH CASES AND CONTROLS. THREE HUNDRED AND NINETEEN DIFFERENTIALLY METHYLATED REGIONS (DMR) WERE IDENTIFIED SPREAD ACROSS THE GENOME. PATHWAY ANALYSIS REVEALED THAT DMRS ASSOCIATED WITH GENES WERE INVOLVED IN EMBRYONIC AND VASCULAR DEVELOPMENT, AND SIGNALLING PATHWAYS SUCH AS TGFBETA AND FGF. EXPRESSION OF TOP DIFFERENTIALLY METHYLATED GENE HOXA5 SHOWED A SIGNIFICANT NEGATIVE CORRELATION WITH DNA METHYLATION. INTERESTINGLY, DNA METHYLATION AGE AND CHRONOLOGICAL AGE WERE HIGHLY CORRELATED, BUT THERE WAS NO EVIDENCE OF ACCELERATED AGE-RELATED DNA METHYLATION IN THE ARTERIES OF CKD PATIENTS. IN CONCLUSION, WE DEMONSTRATED THAT DIFFERENTIAL DNA METHYLATION IN THE ARTERIAL TISSUE OF CKD PATIENTS REPRESENTS A POTENTIAL MEDIATOR OF ARTERIAL PATHOLOGY AND MAY BE USED TO UNCOVER NOVEL PATHWAYS IN THE GENESIS OF CKD-ASSOCIATED COMPLICATIONS. 2021 18 2494 25 EPIGENETICS AND CHRONIC LYMPHOCYTIC LEUKEMIA. THE DNA METHYLATION LEVEL IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA IS GENERALLY LOWER THAN HEALTHY INDIVIDUALS. ALTHOUGH DNA METHYLATION IS GLOBALLY DECREASED, REGIONAL HYPERMETHYLATION OF GENE PROMOTERS LEADS TO GENE SILENCING. MANY OF THESE GENES HAVE TUMOR SUPPRESSOR PHENOTYPES. UNLIKE MUTATIONS OR DELETIONS, HYPERMETHYLATION IS POTENTIALLY REVERSIBLE AFTER INHIBITION WITH DNA METHYLATION MODULATORS. MYELODYSPLASTIC SYNDROME HAS BEEN A MODEL DISEASE IN WHICH TREATMENT OF PATIENTS RESULTS IN DEMETHYLATION OF SPECIFIC GENES. THE STORY IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA IS SLOWLY UNRAVELING AS EPIGENETIC MODIFICATIONS LIKELY ALSO PLAY AN IMPORTANT ROLE. ONGOING CLINICAL TRIALS CORRELATING CLINICAL RESPONSE TO GENE EXPRESSION AFTER TREATMENT WITH DNA METHYLATION INHIBITORS WILL ULTIMATELY ALLOW US TO BETTER RISK STRATIFY AND PREDICT THE SUBGROUP OF PATIENTS WHO WILL BENEFIT FROM TREATMENT WITH THIS CLASS OF DRUGS. 2006 19 2400 32 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 20 6723 42 VITAMIN D RECEPTOR GENE METHYLATION IN HEPATOCELLULAR CARCINOMA. WORLDWIDE, HEPATOCELLULAR CARCINOMA (HCC) IS THE MAJOR SUBTYPE OF PRIMARY LIVER CANCERS. HCC IS TYPICALLY DIAGNOSED LATE IN ITS COURSE. WITH RESPECT TO CANCER, THE GENOMIC ACTIONS OF VITAMIN D ARE MEDIATED THROUGH BINDING TO THE VITAMIN D RECEPTOR (VDR), WHICH ALLOWS IT TO MODULATE THE EXPRESSION OF GENES IN A CELL-AND TISSUE-SPECIFIC MANNER. EPIGENETICS IS A RAPIDLY EVOLVING FIELD OF GENETIC STUDY APPLICABLE TO HCC. CHANGES IN DNA METHYLATION PATTERNS ARE THOUGHT TO BE EARLY EVENTS IN HEPATOCARCINOGENESIS. CURCUMIN HAS GREAT POTENTIAL AS AN EPIGENETIC AGENT. ACCORDINGLY, THE CURRENT STUDY HAS BEEN DESIGNED TO STUDY THE METHYLATION STATUS OF VDR GENE PROMOTER FOR THE FIRST TIME IN HCC AIMING TO FIND ITS CLINICAL SIGNIFICANCE AND POTENTIAL SCREENING ROLE IN CHRONIC LIVER DISEASE (CLD). ADDITIONALLY, WE AIMED TO INVESTIGATE, THE EFFECT OF CURCUMIN ON HCC CELL LINE, AIMING TO DISCOVER NEW THERAPEUTIC TARGETS THROUGH EPIGENETICS. THIS STUDY WAS CONDUCTED ON 45 FORMALIN-FIXED, PARAFFIN-EMBEDDED LIVER TISSUE BLOCKS INCLUDING 15 HCC SAMPLES (GROUP A), 15 CLD SAMPLES (GROUP B) AND 15 APPARENTLY NORMAL TISSUE TAKEN FROM AROUND BENIGN LESIONS (GROUP C). METHYLATION SPECIFIC RESTRICTION DIGESTION AND QPCR WERE DONE ON ALL SAMPLES AFTER DNA EXTRACTION. THE PERCENTAGE OF VDR GENE PROMOTER METHYLATION WAS SIGNIFICANTLY HIGHER IN THE HCC GROUP COMPARED TO BOTH CLD AND CONTROL GROUPS (P < 0.01). VDR PROMOTER METHYLATION BY (MS-QPCR) WAS DECREASED AND THE RELATIVE EXPRESSION OF VDR BY (QRT-PCR) WAS MARKEDLY INCREASED IN A DOSE-DEPENDENT FASHION IN CELLS GROWN IN CURCUMIN-ADEQUATE MEDIUM. IN CONCLUSION, THIS STUDY MAY OPEN A NEW GATE FOR THE USE OF VDR PROMOTER METHYLATION AS A POTENTIAL BIOMARKER IN HCC. 2018