A systematic comparison of quantitative high-resolution DNA methylation analysis and methylation-specific PCR

Assessment of DNA methylation has become a critical factor for the identification, development and application of methylation based biomarkers. Here we describe a systematic comparison of a quantitative high-resolution mass spectrometry-based approach (MassARRAY), pyrosequencing and the broadly used methylation-specific PCR (MSP) technique analyzing clinically relevant epigenetically silenced genes in acute myeloid leukemia (AML). By MassARRAY and pyrosequencing, we identified significant DNA methylation differences at the ID4 gene promoter and in the 5′ region of members of the SFRP gene family in 62 AML patients compared with healthy controls. We found a good correlation between data obtained by MassARRAY and pyrosequencing (correlation coefficient R² = 0.88). MSP-based assessment of the identical samples showed less pronounced differences between AML patients and controls. By direct comparison of MSP-derived and MassARRAY-based methylation data as well as pyrosequencing, we could determine overestimation of DNA methylation data by MSP. We found sequence-context dependent highly variable cut-off values of quantitative DNA methylation values serving as discriminator for the two MSP methylation categories. Moreover, good agreements between quantitative methods and MSP could not be achieved for all investigated loci. Significant correlation of the quantitative assessment but not of MSP-derived methylation data with clinically important characteristics in our patient cohort demonstrated clinical relevance of quantitative DNA methylation assessment. Taken together, while MSP is still the most commonly applied technique for DNA methylation assessment, our data highlight advantages of quantitative approaches for precise characterization and reliable biomarker use of aberrant DNA methylation in primary patient samples, particularly.     

Identification of differentially methylated markers among cytogenetic risk groups of acute myeloid leukemia

Aberrant DNA methylation is known to occur in cancer, including hematological malignancies such as acute myeloid leukemia (AML). However, less is known about whether specific methylation profiles characterize specific subcategories of AML. We examined this issue by using comprehensive high-throughput array-based relative methylation analysis (CHARM) to compare methylation profiles among patients in different AML cytogenetic risk groups. We found distinct profiles in each group, with the high-risk group showing overall increased methylation compared with low- and mid-risk groups. The differentially methylated regions (DMRs) distinguishing cytogenetic risk groups of AML were enriched in the CpG island shores. Specific risk-group associated DMRs were located near genes previously known to play a role in AML or other malignancies, such as MN1, UHRF1, HOXB3, and HOXB4, as well as TRIM71, the function of which in cancer is not well characterized. These findings were verified by quantitative bisulfite pyrosequencing and by comparison with results available at the TCGA cancer genome browser. To explore the potential biological significance of the observed methylation changes, we correlated our findings with gene expression data available through the TCGA database. The results showed that decreased methylation at HOXB3 and HOXB4 was associated with increased gene expression of both HOXB genes specific to the mid-risk AML, while increased DNA methylation at DCC distinctive to the high-risk AML was associated with increased gene expression. Our results suggest that the differential impact of cytogenetic changes on AML prognosis may, in part, be mediated by changes in methylation.     

Assessing combined methylation–sensitive high resolution melting and pyrosequencing for the analysis of heterogeneous DNA methylation

Heterogeneous DNA methylation leads to difficulties in accurate detection and quantification of methylation. Methylation-sensitive high resolution melting (MS-HRM) is unique among regularly used methods for DNA methylation analysis in that heterogeneous methylation can be readily identified, although not quantified, by inspection of the melting curves. Bisulfite pyrosequencing has been used to estimate the level of heterogeneous methylation by quantifying methylation levels present at individual CpG dinucleotides. Sequentially combining the two methodologies using MS-HRM to screen the amplification products prior to bisulfite pyrosequencing would be advantageous. This would not only replace the quality control step using agarose gel analysis prior to the pyrosequencing step but would also provide important qualitative information in its own right. We chose to analyze DAPK1 as it is an important tumor suppressor gene frequently heterogeneously methylated in a number of malignancies, including chronic lymphocytic leukemia (CLL). A region of the DAPK1 promoter was analyzed in ten CLL samples by MS-HRM. By using a biotinylated primer, bisulfite pyrosequencing could be used to directly analyze the samples. MS-HRM revealed the presence of various extents of heterogeneous DAPK1 methylation in all CLL samples. Further analysis of the biotinylated MS-HRM products by bisulfite pyrosequencing provided quantitative information for each CpG dinucleotide analyzed, and confirmed the presence of heterogeneous DNA methylation. Whereas each method could be used individually, MS-HRM and bisulfite pyrosequencing provided complementary information for the assessment of heterogeneous methylation.     

Comparative analysis of methods to determine DNA methylation levels of a tumor-related microRNA gene

Quantifying levels of DNA methylation in tumors is a useful approach for the identification of potential tumor suppressors and to find biomarkers that can be used as prognostic or therapeutic indicators. In the current study, we compared three methods commonly used for quantifying DNA methylation—bisulfite pyrosequencing, quantitative methylation-specific PCR (Q-MSP), and MethyLight—by focusing on the CpG island of the gene encoding the microRNA-34b and microRNA-34c (miR-34b/c); aberrant regulation of this miR is associated with various human malignancies, including gastric cancer. Standard curve analysis using control DNA samples demonstrated the highest quantitative accuracy in Q-MSP analysis. We also carried out methylation analysis using gastric mucosa specimens obtained from gastric cancer patients. We found a high correlation between methylation levels determined by Q-MSP and those determined by MethyLight (R² = 0.952), whereas the results of bisulfite pyrosequencing and the other two methods were less well correlated (R² = 0.864 and R² = 0.804 for Q-MSP and MethyLight, respectively). This may reflect possible PCR bias in the pyrosequencing technique, which we show can be corrected for by applying a cubic approximate equation to the original data. Thus, although results obtained by the different DNA methylation analysis techniques are largely comparable, an appropriate correction may be necessary for stringent comparison.     

Methylation enrichment pyrosequencing: combining the specificity of MSP with validation by pyrosequencing

It has been suggested that detection of aberrant DNA methylation in clinical specimens such as sputum or saliva may be a valuable tumour biomarker. Any clinically applicable detection technique must combine high sensitivity with high specificity. In this study we describe methylation enrichment pyrosequencing (MEP), which benefits from the high sensitivity and specificity of methylation-specific PCR (MSP) but has a second, confirmatory, pyrosequencing step. The pyrosequencing reaction is rapid, relatively inexpensive and offers significant logistical advantages over previously described validation methods. As proof of principle, we illustrate MEP using assays of p16 and cyclin A1 promoters in a methylated DNA dilution matrix and also in a clinical setting using paired saliva and oral tumour specimens. Our results confirm that mis-priming of MSP, with subsequent false positive results, can occur frequently (perhaps 10%) in assays combining high numbers of PCR cycles and low concentrations of starting DNA. In our clinical example, MEP of saliva-derived DNA was more sensitive than standard non-methylation-specific pyrosequencing as illustrated using p16 and cyclin A1 promoter methylation assays.     

Assessing combined methylation-sensitive high resolution melting and pyrosequencing for the analysis of heterogeneous DNA methylation

Heterogeneous DNA methylation leads to difficulties in accurate detection and quantification of methylation. Methylation-sensitive high resolution melting (MS-HRM) is unique among regularly used methods for DNA methylation analysis in that heterogeneous methylation can be readily identified, although not quantified, by inspection of the melting curves. Bisulfite pyrosequencing has been used to estimate the level of heterogeneous methylation by quantifying methylation levels present at individual CpG dinucleotides. Sequentially combining the two methodologies using MS-HRM to screen the amplification products prior to bisulfite pyrosequencing would be advantageous. This would not only replace the quality control step using agarose gel analysis prior to the pyrosequencing step but would also provide important qualitative information in its own right. We chose to analyze DAPK1 as it is an important tumor suppressor gene frequently heterogeneously methylated in a number of malignancies, including chronic lymphocytic leukemia (CLL). A region of the DAPK1 promoter was analyzed in ten CLL samples by MS-HRM. By using a biotinylated primer, bisulfite pyrosequencing could be used to directly analyze the samples. MS-HRM revealed the presence of various extents of heterogeneous DAPK1 methylation in all CLL samples. Further analysis of the biotinylated MS-HRM products by bisulfite pyrosequencing provided quantitative information for each CpG dinucleotide analyzed, and confirmed the presence of heterogeneous DNA methylation. Whereas each method could be used individually, MS-HRM and bisulfite pyrosequencing provided complementary information for the assessment of heterogeneous methylation.Key words: MS-HRM, pyrosequencing, digital PCR, heterogeneous DNA methylation, DAPK1, chronic lymphocytic leukemia     

DNA hypermethylation and DNA hypomethylation is present at different loci in chronic kidney disease

Genetic risk factors for chronic kidney disease (CKD) are being identified through international collaborations. By comparison, epigenetic risk factors for CKD have only recently been considered using population-based approaches. DNA methylation is a major epigenetic modification that is associated with complex diseases, so we investigated methylome-wide loci for association with CKD. A total of 485,577 unique features were evaluated in 255 individuals with CKD (cases) and 152 individuals without evidence of renal disease (controls). Following stringent quality control, raw data were quantile normalized and β values calculated to reflect the methylation status at each site. The difference in methylation status was evaluated between cases and controls with resultant P values adjusted for multiple testing. Genes with significantly increased and decreased levels of DNA methylation were considered for biological relevance by functional enrichment analysis using KEGG pathways in Partek Genomics Suite. Twenty-three genes, where more than one CpG per loci was identified with P_adjusted < 10^?8, demonstrated significant methylation changes associated with CKD and additional support for these associated loci was sought from published literature. Strong biological candidates for CKD that showed statistically significant differential methylation include CUX1, ELMO1, FKBP5, INHBA-AS1, PTPRN2, and PRKAG2 genes; several genes are differentially methylated in kidney tissue and RNA-seq supports a functional role for differential methylation in ELMO1 and PRKAG2 genes. This study reports the largest, most comprehensive, genome-wide quantitative evaluation of DNA methylation for association with CKD. Evidence confirming methylation sites influence development of CKD would stimulate research to identify epigenetic therapies that might be clinically useful for CKD.     

Prognostic value of three different methods of MGMT promoter methylation analysis in a prospective trial on newly diagnosed glioblastoma

Hypermethylation in the promoter region of the MGMT gene encoding the DNA repair protein O⁶-methylguanine-DNA methyltransferase is among the most important prognostic factors for patients with glioblastoma and predicts response to treatment with alkylating agents like temozolomide. Hence, the MGMT status is widely determined in most clinical trials and frequently requested in routine diagnostics of glioblastoma. Since various different techniques are available for MGMT promoter methylation analysis, a generally accepted consensus as to the most suitable diagnostic method remains an unmet need. Here, we assessed methylation-specific polymerase chain reaction (MSP) as a qualitative and semi-quantitative method, pyrosequencing (PSQ) as a quantitative method, and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) as a semi-quantitative method in a series of 35 formalin-fixed, paraffin-embedded glioblastoma tissues derived from patients treated in a prospective clinical phase II trial that tested up-front chemoradiotherapy with dose-intensified temozolomide (UKT-05). Our goal was to determine which of these three diagnostic methods provides the most accurate prediction of progression-free survival (PFS). The MGMT promoter methylation status was assessable by each method in almost all cases (n = 33/35 for MSP; n = 35/35 for PSQ; n = 34/35 for MS-MLPA). We were able to calculate significant cut-points for the continuous methylation signals at each CpG site analysed by PSQ (range, 11.5 to 44.9%) and at one CpG site assessed by MS-MLPA (3.6%) indicating that a dichotomisation of continuous methylation data as a prerequisite for comparative survival analyses is feasible. Our results show that, unlike MS-MLPA, MSP and PSQ provide a significant improvement of predicting PFS compared with established clinical prognostic factors alone (likelihood ratio tests: p<0.001). Conclusively, taking into consideration prognostic value, cost effectiveness and ease of use, we recommend pyrosequencing for analyses of MGMT promoter methylation in high-throughput settings and MSP for clinical routine diagnostics with low sample numbers.     

Maternal cigarette smoking during pregnancy and offspring DNA methylation in midlife

Maternal smoking in pregnancy (MSP) has been associated with DNA methylation in specific CpG sites (CpGs) in infants and children. We investigated whether MSP, independent of own personal active smoking, was associated with midlife DNA methylation in CpGs that were previously identified in studies of MSP-DNA methylation in children. We used data on MSP collected from pregnant mothers of 89 adult women born in 1959–1964 and measured DNA methylation in blood (granulocytes) collected in 2001–2007 (mean age: 43 years). Seventeen CpGs were differentially methylated by MSP, with multiple CpGs mapping to CYP1A1, MYO1G, AHRR, and GFI1. These associations were consistent in direction with prior studies (e.g., MSP associated with more and less methylation in AHRR and CYP1A1, respectively) and, with the exception of AHRR CpGs, were not substantially altered by adjustment for active smoking. These preliminary results confirm prior prospective reports that MSP influences the offspring DNA methylation, and extends the timeframe to midlife, and suggest that these effects may persist into adulthood, independently of active smoking.     

Genome-wide DNA methylation profiling in rheumatoid arthritis identifies disease-associated methylation changes that are distinct to individual T- and B-lymphocyte populations

Changes to the DNA methylome have been described in patients with rheumatoid arthritis (RA). In previous work, we reported genome-wide methylation differences in T-lymphocyte and B-lymphocyte populations from healthy individuals. Now, using HumanMethylation450 BeadChips to interrogate genome-wide DNA methylation, we have determined disease-associated methylation changes in blood-derived T- and B-lymphocyte populations from 12 female patients with seropositive established RA, relative to 12 matched healthy individuals. Array data were analyzed using NIMBL software and bisulfite pyrosequencing was used to validate array candidates. Genome-wide DNA methylation, determined by analysis of LINE-1 sequences, revealed higher methylation in B-lymphocytes compared with T-lymphocytes (P ≤ 0.01), which is consistent with our findings in healthy individuals. Moreover, loci-specific methylation differences that distinguished T-lymphocytes from B-lymphocytes in healthy individuals were also apparent in RA patients. However, disease-associated methylation differences were also identified in RA. In these cases, we identified 509 and 252 CpGs in RA-derived T- and B-lymphocytes, respectively, that showed significant changes in methylation compared with their cognate healthy counterparts. Moreover, this included a restricted set of 32 CpGs in T-lymphocytes and 20 CpGs in B-lymphocytes (representing 15 and 10 genes, respectively, and including two, MGMT and CCS, that were common to both cell types) that displayed more substantial changes in methylation. These changes, apparent as hyper- or hypo-methylation, were independently confirmed by pyrosequencing analysis. Validation by pyrosequencing also revealed additional sites in some candidate genes that also displayed altered methylation in RA. In this first study of genome-wide DNA methylation in individual T- and B-lymphocyte populations in RA patients, we report disease-associated methylation changes that are distinct to each cell type and which support a role for discrete epigenetic regulation in this disease.     

Genome wide analysis of acute myeloid leukemia reveal leukemia specific methylome and subtype specific hypomethylation of repeats

Methylated DNA immunoprecipitation followed by high-throughput sequencing (MeDIP-seq) has the potential to identify changes in DNA methylation important in cancer development. In order to understand the role of epigenetic modulation in the development of acute myeloid leukemia (AML) we have applied MeDIP-seq to the DNA of 12 AML patients and 4 normal bone marrows. This analysis revealed leukemia-associated differentially methylated regions that included gene promoters, gene bodies, CpG islands and CpG island shores. Two genes (SPHKAP and DPP6) with significantly methylated promoters were of interest and further analysis of their expression showed them to be repressed in AML. We also demonstrated considerable cytogenetic subtype specificity in the methylomes affecting different genomic features. Significantly distinct patterns of hypomethylation of certain interspersed repeat elements were associated with cytogenetic subtypes. The methylation patterns of members of the SINE family tightly clustered all leukemic patients with an enrichment of Alu repeats with a high CpG density (P<0.0001). We were able to demonstrate significant inverse correlation between intragenic interspersed repeat sequence methylation and gene expression with SINEs showing the strongest inverse correlation (R(2) = 0.7). We conclude that the alterations in DNA methylation that accompany the development of AML affect not only the promoters, but also the non-promoter genomic features, with significant demethylation of certain interspersed repeat DNA elements being associated with AML cytogenetic subtypes. MeDIP-seq data were validated using bisulfite pyrosequencing and the Infinium array.     

Comparison of bisulfite sequencing PCR with pyrosequencing for measuring differences in DNA methylation

DNA methylation strongly affects chromatin structure and the regulation of gene expression. For many years, bisulfite sequencing PCR (BSP) has served as the “gold standard” for measuring DNA methylation. However, with the evolution of pyrosequencing as a tool to evaluate DNA methylation, the need arises to compare the relative efficiencies of the two techniques in measuring DNA methylation. We provide for the first time a direct assessment of BSP and pyrosequencing to detect and quantify hypomethylation, hypermethylation, and mixed methylation of the ABCB1 promoter in various drug-sensitive and drug-resistant MCF-7 breast cancer cell lines through head-to-head experimentation. Our findings indicate that although both methods can reliably detect increased, decreased, and mixed methylation of DNA, BSP appears to be more sensitive than pyrosequencing at detecting strong hypermethylation of DNA. However, we also observed greater variability in the methylation of CpG sites by BSP, possibly due to the additional bacterial cloning step required by BSP over pyrosequencing. BSP and pyrosequencing equally detected hypomethylation and mixed methylation of DNA. The ability of pyrosequencing to reliably detect differences in DNA methylation across cell populations without requiring the cloning of bisulfite-treated DNA into bacterial expression vectors was seen as a major advantage of this technique.     

Increased DNA methylation of neuropsychiatric genes occurs in borderline personality disorder

Borderline personality disorder (BPD) is a complex psychiatric disease of increasing importance. Epigenetic alterations are hallmarks for altered gene expression and could be involved in the etiology of BPD. In our study we analyzed DNA methylation patterns of 14 neuropsychiatric genes (COMT, DAT1, GABRA1, GNB3, GRIN2B, HTR1B, HTR2A, 5-HTT, MAOA, MAOB, NOS1, NR3C1, TPH1 and TH). DNA methylation was analyzed by bisulfite restriction analysis and pyrosequencing in whole blood samples of patients diagnosed with DSM-IV BPD and in controls. Aberrant methylation was not detectable using bisulfite restriction analysis, but a significantly increased methylation of HTR2A, NR3C1, MAOA, MAOB and soluble COMT (S-COMT) was revealed for BPD patients using pyrosequencing. For HTR2A the average methylation of four CpG sites was 0.8% higher in BPD patients compared to controls (p = 0.002). The average methylation of NR3C1 was 1.8% increased in BPD patients compared to controls (p = 0.0003) and was higher at 2 out of 8 CpGs (p ≤ 0.04). In females, an increased average methylation (1.5%) of MAOA was observed in BPD patients compared to controls (p = 0.046). A similar trend (1.4% higher methylation) was observed for MAOB in female BPD patients and increased methylation was significant for 1 out of 6 CpG sites. For S-COMT, a higher methylation of 2 out of 4 CpG sites was revealed in BPD patients (p ≤ 0.02). In summary, methylation signatures of several promoter regions were established and a significant increased average methylation (1.7%) occurred in blood samples of BPD patients (p < 0.0001). Our data suggest that aberrant epigenetic regulation of neuropsychiatric genes may contribute to the pathogenesis of BPD.     

A comparison of existing global DNA methylation assays to low-coverage whole-genome bisulfite sequencing for epidemiological studies

DNA methylation is an epigenetic mark at the interface of genetic and environmental factors relevant to human disease. Quantitative assessments of global DNA methylation levels have therefore become important tools in epidemiology research, particularly for understanding effects of environmental exposures in complex diseases. Among the available methods of quantitative DNA methylation measurements, bisulfite sequencing is considered the gold standard, but whole-genome bisulfite sequencing (WGBS) has previously been considered too costly for epidemiology studies with high sample numbers. Pyrosequencing of repetitive sequences within bisulfite-treated DNA has been routinely used as a surrogate for global DNA methylation, but a comparison of pyrosequencing to WGBS for accuracy and reproducibility of methylation levels has not been performed. This study compared the global methylation levels measured from uniquely mappable (non-repetitive) WGBS sequences to pyrosequencing assays of several repeat sequences and repeat assay-matched WGBS data and determined uniquely mappable WGBS data to be the most reproducible and accurate measurement of global DNA methylation levels. We determined sources of variation in repetitive pyrosequencing assays to be PCR amplification bias, PCR primer selection bias in methylation levels of targeted sequences, and inherent variability in methylation levels of repeat sequences. Low-coverage, uniquely mappable WGBS showed the strongest correlation between replicates of all assays. By using multiplexing by indexed bar codes, the cost of WGBS can be lowered significantly to improve the accuracy of global DNA methylation assessments for human studies.     

Detection of methylated tissue factor pathway inhibitor 2 and human long DNA in fecal samples of patients with colorectal cancer in China

Background: To investigate the feasibility of detecting methylated tissue factor pathway inhibitor (TFPI2) and quantifying human long DNA with fluorescent quantitative Alu PCR in fecal DNA as a non-invasive screening tool for colorectal cancer (CRC). Materials and Methods: Methylation-specific PCR (MSP) was performed to analyze TFPI2 gene promoter methylation status in a blinded fashion in stool samples taken from 30 endoscopically diagnosed healthy controls, 20 patients with adenomas, and 60 patients with colorectal cancer. Real-time Alu PCR was used to quantify human long DNA. Results: The specificity of fecal TFPI2 MSP assay and long DNA assay was 100% and 83.3%, respectively. The sensitivity of fecal TFPI2 MSP assay and long DNA assay was 68.3% and 53.3%, respectively. The sensitivity of fecal DNA assay (either marker being positive) was 86.7%, which was high for CRC. Conclusions: Our results have demonstrated the feasibility of using TFPI2 methylation and quantify human long DNA with fluorescent quantitative Alu PCR in fecal samples as a new noninvasive test for CRC.     

Systematic cross-validation of 454 sequencing and pyrosequencing for the exact quantification of DNA methylation patterns with single CpG resolution

Background  

New high-throughput sequencing technologies promise a very sensitive and high-resolution analysis of DNA methylation patterns in quantitative terms. However, a detailed and comprehensive comparison with existing validated DNA methylation analysis methods is not yet available. Therefore, a systematic cross-validation of 454 sequencing and conventional pyrosequencing, both of which offer exact quantification of methylation levels with a single CpG dinucleotide resolution, was performed.     

Quality control and statistical modeling for environmental epigenetics: A study on in utero lead exposure and DNA methylation at birth

DNA methylation data assayed using pyrosequencing techniques are increasingly being used in human cohort studies to investigate associations between epigenetic modifications at candidate genes and exposures to environmental toxicants and to examine environmentally-induced epigenetic alterations as a mechanism underlying observed toxicant-health outcome associations. For instance, in utero lead (Pb) exposure is a neurodevelopmental toxicant of global concern that has also been linked to altered growth in human epidemiological cohorts; a potential mechanism of this association is through alteration of DNA methylation (e.g., at growth-related genes). However, because the associations between toxicants and DNA methylation might be weak, using appropriate quality control and statistical methods is important to increase reliability and power of such studies. Using a simulation study, we compared potential approaches to estimate toxicant-DNA methylation associations that varied by how methylation data were analyzed (repeated measures vs. averaging all CpG sites) and by method to adjust for batch effects (batch controls vs. random effects). We demonstrate that correcting for batch effects using plate controls yields unbiased associations, and that explicitly modeling the CpG site-specific variances and correlations among CpG sites increases statistical power. Using the recommended approaches, we examined the association between DNA methylation (in LINE-1 and growth related genes IGF2, H19 and HSD11B2) and 3 biomarkers of Pb exposure (Pb concentrations in umbilical cord blood, maternal tibia, and maternal patella), among mother-infant pairs of the Early Life Exposures in Mexico to Environmental Toxicants (ELEMENT) cohort (n = 247). Those with 10 μg/g higher patella Pb had, on average, 0.61% higher IGF2 methylation (P = 0.05). Sex-specific trends between Pb and DNA methylation (P < 0.1) were observed among girls including a 0.23% increase in HSD11B2 methylation with 10 μg/g higher patella Pb.     

Effect of alcohol consumption on peripheral blood Alu methylation in Korean men

Alcohol use disorders (AUD) are defined as alcohol abuse and alcohol dependence, which create a substantial public health problem worldwide. To date, no therapeutic can effectively solve these problems. They are complex diseases characterized by both genetic and environmental factors. DNA methylation can act as a downstream effector of environmental signals and account for multi-factorial nature of the disease. Global DNA methylation of peripheral blood cells has recently been proposed as a potential biomarker for disease risk. Alu elements host one-quarter of CpG dinucelotides in the genome to function as proxies for global DNA methylation. In this study, we evaluated the Alu methylation in the peripheral blood DNA of healthy volunteers and AUD patients using the pyrosequencing technology. The Alu methylation level is significantly higher in AUD compared to healthy controls (23.4?±?1.6 versus 22.1?±?1.0, t?=?7.83, p?<?0.0001). Moreover, significant correlation was found between Alu methylation and alcohol use disorders identification test score (r?=?0.250, p?<?0.0001), alcohol problem (r?=?0.294, p?<?0.0001), and life position (r?=?–0.205, p?=?0.0005). Overall, these novel findings indicate that alcohol-related increase in Alu methylation might play a complex role in the etiology and pathogenesis of AUD. Further studies are required to elucidate the mechanisms underlying this relationship.