Methylation Analysis to Detect CIN3+ in High-Risk Human Papillomavirus-Positive Self-Samples From the Population-Based Cervical Cancer Screening Program.

Since 2017, a self-sampling device has been introduced to the Dutch population-based screening program to enable higher participation rates. However, routine triage cytology cannot be performed on self-sampling material. Methylation is an alternative triage method that can be performed directly on DNA extracted from self-samples. Recently, we tested a set of 15 published cervical intraepithelial neoplasia grade 3 or worse (CIN3+)-specific methylation markers and found a panel of 3 markers with a sensitivity of 82% and a specificity of 74%. In this study, we determined the sensitivity and specificity of 2 commercial assays using quantitative methylation-specific PCR. DNA from the same cohort of high-risk human papillomavirus-positive self-sampled material obtained through the population-based screening program in the North of the Netherlands from women with CIN2 or less (

DNA methylation markers as triage test for the early identification of cervical lesions in a Chinese population.

Objective strategies are required in cervical cancer screening. We have identified several DNA methylation markers with high sensitivity and specificity to detect cervical intraepithelial neoplasia 2 or worse (CIN2+) in Dutch women. Our study aims to analyze the diagnostic characteristics of these markers in a Chinese cohort. A total of 246 liquid-based cytology samples were included, of which 205 women underwent colposcopy due to an abnormal cytology result (atypical squamous cells of undetermined significance [ASCUS] or worse), while 227 were tested high-risk human papillomavirus (hrHPV) positive. All six individual markers (ANKRD18CP, C13ORF18, EPB41L3, JAM3, SOX1 and ZSCAN1) showed enhanced methylation levels and frequency with increasing severity of the underlying lesion (P ≤ .001). In cytological abnormal women, sensitivity to detect CIN2+ was 79%, 76% and 72% for the three panels (C13ORF18/EBP41L3/JAM3, C13ORF18/ANKRD18CP/JAM3 and ZSCAN1/SOX1, respectively), with a specificity of 57%, 65% and 68%. For the first two panels, these diagnostic characteristics were similar to the Dutch cohort, while for ZSCAN1/SOX1 the sensitivity was higher in the Chinese cohort, but with a lower specificity (both P < .05). In hrHPV-positive samples, similar sensitivity and specificity for the detection of CIN2+ were found as for the abnormal cytology cohort, which were now all similar between both cohorts and non-inferior to HPV16/18 genotyping. Our analysis reveals that the diagnostic performances are highly comparable for C13ORF18/EBP41L3/JAM3 and C13ORF18/ANKRD18CP/JAM3 methylation marker panels in both Chinese and Dutch cohorts. In conclusion, methylation panels identified in a Dutch population are also applicable for triage testing in cervical cancer screening in China.

SOX14 hypermethylation as a tumour biomarker in cervical cancer.

BACKGROUND: The association between SOX14 and cancer has been reported. The aim of this study was to identify and validate the potential value of SOX14 methylation in the early detection of cervical cancer. METHODS: First, we extracted the data for SOX14 methylation and expression within cervical cancer from The Cancer Genome Atlas (TCGA) database and analysed them via UALCAN, Wanderer, MEXPRESS and LinkedOmics. Subsequently, according to the bioinformatics findings, primers and probes were designed for the most significantly differentiated methylation CpG site and synthesized for methylation-specific PCR (MSP) and quantitative methylation-specific PCR (QMSP) to verify SOX14 methylation in both cervical tissuses and liquid-based cell samples. Eventually, the clinical diagnostic efficacy of SOX14 methylation in the normal, cervical intraepithelial neoplasia, and cancer groups was analysed by ROCAUC. RESULTS: Pooled analysis demonstrated that SOX14 methylation levels were significantly increased in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) compared to normal tissues (P < 0.001). Both the verification and validation cohorts indicated that the methylation level and the positive rate of SOX14 gradually increased with increasing severity from normal to cancer samples (P < 0.01). When the cut-off value was set as 128.45, the sensitivity and specificity of SOX14 hypermethylation in the diagnosis of cervical cancer were 94.12 and 86.46%, respectively. When taken as a screening biomarker (>CINII), the sensitivity was 74.42% and the specificity was 81.48%, with a cut-off value of 10.37. CONCLUSION: SOX14 hypermethylation is associated with cervical cancer and has the potential to be a molecular biomarker for the screening and early diagnosis of cervical cancer.

TFPI2 and NDRG4 gene promoter methylation analysis in peripheral blood mononuclear cells are novel epigenetic noninvasive biomarkers for colorectal cancer diagnosis.

BACKGROUND: As a result of the growing prevalence of colorectal cancer (CRC), new screening and early detection methods are required. Among the novel biomarkers, DNA methylation has emerged as a high-potential diagnosis/screening molecular marker. The present study aimed to assess non-invasive early diagnosis of CRC by examining promoter methylation of TFPI2 and NDRG4 genes in peripheral blood mononuclear cells (PBMCs). METHODS: Fifty CRC patients and 50 normal controls were recruited to the present study. Quantitative methylation of the promoter region of the TFPI2 and NDRG4 genes was analyzed in DNA extracted from PBMCs of all cases and control subjects using a methylation-quantification endonuclease-resistant DNA (MethyQESD) method. RESULTS: The sensitivity and specificity of the TFPI2 gene for the diagnosis of CRC was 88% and 92%, respectively, and, for the NDRG4 gene, it was 86% and 92%, respectively. The methylation range for the TFPI2 gene was 43.93% and 11.56% in patients and controls, respectively, and, for the NDRG4 gene, it was 38.8% in CRC patients and 12.23% in healthy controls (p < 0.001). In addition, we observed that a higher percentage of methylation was correlated with the higher stage of CRC. CONCLUSIONS: The results of the present study reveal that PBMCs are reliable sources of methylation analysis for CRC screening. Furthermore, the TFPI2 and NDRG4 genes provide sufficiently high sensitivity and specificity to be nominated for use in a novel noninvasive CRC screening method in PBMCs.

SMYD3 promoter hypomethylation is associated with the risk of colorectal cancer.

AIM: SMYD3 encodes histone lysine methyltransferase. The goal of our study was to investigate the association between SMYD3 methylation and colorectal cancer (CRC). MATERIALS & METHODS: SMYD3 methylation was measured by quantitative methylation-specific PCR method in 117 pairs of CRC tumor and para-tumor tissues. RESULTS: Significantly lower SMYD3 methylation was observed in CRC tumor tissues than para-tumor tissues (p = 0.002). Further subgroup analysis by clinical features showed that significantly lower SMYD3 methylation were only observed in the CRC patients with tumors of moderately and well differentiation, positive lymph node metastasis, and stage III + IV. CONCLUSION: Our work reported for the first time that SMYD3 promoter hypomethylation was associated with CRC.

Significant association of PRMT6 hypomethylation with colorectal cancer.

BACKGROUND: Protein arginine N-methyltransferase 6 (PRMT6) was deemed to be indispensable in the variety of biological processes. Upregulated PRMT6 was found in various human diseases including cancer. Herein, we investigated the performance of PRMT6 methylation in the diagnosis for CRC. METHODS: A quantitative methylation-specific polymerase chain reaction (qMSP) method was used to measure PRMT6 promoter methylation. The percentage of methylated reference (PMR) was applied to represent gene methylation level. RESULTS: Our data indicated that PRMT6 promoter methylation levels were significantly lower in CRC tissues than those in paired nontumor tissues (median PMR: 36.93% vs 63.12%, P = 1E-6) and normal intestinal tissues (median PMR: 36.93% vs 506.55%, P = 8E-12). We further examined the potential role of PRMT6 hypomethylation by the receiver operating characteristic (ROC) curve. Our results showed that the area under the curve (AUC) was 0.644 (95% CI = 0.596-0.733) between CRC tissues and paired nontumor tissues, 0.958 (95% CI = 0.919-0.998) between CRC tissues and normal intestinal tissues, and 0.899 (95% CI = 0.825-0.972) between paired nontumor tissues and normal intestinal tissues. CONCLUSION: Our study firstly indicated that the hypomethylation of PRMT6 promoter could be a novel diagnostic biomarker for CRC.

Down-regulation of thyroid hormone receptor ß1 gene expression in gastric cancer involves promoter methylation.

Hypermethylation has been shown in the promoter region of the thyroid hormone receptor ß1 (TRß1) gene in several human tumors. However, its role in gastric cancer formation is still unclear. In the study, we analyzed mRNA expression of TRß1 gene using real-time quantitative PCR (qPCR). A quantitative methylation-specific PCR (Q-MSP) assay was used to determine the methylation status of the TRß1 gene promoter region in 46 pair-matched gastric neoplastic and adjacent non-neoplastic tissues. The results showed that TRß1 mRNA expression was significantly reduced in gastric cancer specimens. The methylation of promoter of TRß1 gene in gastric cancer tissues was significantly higher than in adjacent normal tissues. Promoter hypermethylation of the TRß1 gene correlated with tumor infiltration, lymph node metastasis, and distant metastasis, but it was not associated with other clinicopathological characteristics. We treated gastric cancer cell lines MKN-45, MKN-28, SGC-7901, NCI-N87, and SNU-1 with 5-Aza-2-deoxycytidine (5-Aza-dC). The results showed the expression of TRß1 mRNA was increased in MKN-45, MKN-28, SGC-7901, but not increased in NCI-N87 and SNU-1. These results suggest that the TRß1 gene plays important roles in the development of gastric cancer partially through epigenetic mechanisms.

Methylation pattern of methylene tetrahydrofolate reductase and small nuclear ribonucleoprotein polypeptide N promoters in oligoasthenospermia: a case-control study.

Alterations in DNA methylation patterns in several genes may lead to abnormal male sexual development and infertility. This study investigated the promoter methylation status of MTHFR and SNRPN in infertile men from Romania by quantitative methylation-specific PCR in order to investigate possible correlations with sperm abnormalities. The study groups included patients (n=27) with a median age of 31 years (range 26-41 years) as well as controls (n=11) with a median age of 30 years (range 24-37 years) recruited from couples seeking advice for infertility. DNA was isolated from sperm samples and promoter methylation was assessed using direct. Significant trends were detected for both genes that indicate a tendency towards promoter hypermethylation in spermatozoa with low motility (MTHFR P=0.0032, r=0.23; SNRPN P=0.0003, r=0.32) and poor morphology (MTHFR P=0.0012, r=0.27; SNRPN P=0.0003, r=0.33) but no trend was found in cases of low sperm count (MTHFR r=0.007; SNRPN r=0.06). The data indicate that the methylation patterns of the promoters of MTHFR and SNRPN are associated with changes in sperm motility and morphology, which could lead to male infertility. A large number of studies are now focused on the causes of male infertility. Among these are epigenetic modifications, which are important contributors to reproductive pathology in the male by providing dynamic changes of the phenotype according to the environmental and metabolic factors. The most known epigenetic modification is DNA methylation and alterations in this pattern in several genes could induce male infertility. The present study aims to investigate the promoter methylation status of the genes for methylene tetrahydrofolate reductase (MTHFR) and small nuclear ribonucleoprotein polypeptide N (SNRPN) in infertile males from Romania, in order to establish a correlation with sperm parameters. MTHFR is an enzyme involved in the folate pathway and in de novo nucleotide biosynthesis but also a good example for gene-environment interaction in phenotype development. SNRPN is involved in both somatic cell expression and inheritance of the imprint and the methylation pattern of its gene seems to correlate not only with imprinted disorders but also with infertility. Our study includes patients (n=27, median age 31 years, range 26-41 years) recruited from men seeking advice for couple infertility and control group (n=11, median age 30.5 years, range 24-37 years). The data we obtained indicated significant correlations between hypermethylation of the investigated genes and sperm motility and morphology. No significant correlation between DNA methylation and sperm number was found. Our data suggest that methylation pattern of MTHFR and SNRPN is linked with sperm anomalies of motility and morphology and therefore male infertility.

Evaluation of silica spin­column and magnetic bead formats for rapid DNA methylation analysis in clinical and point­of­care settings.

Late-stage cancers lack effective treatment, underscoring the need for early diagnosis to improve prognosis and decrease mortality rates. Molecular markers, such as DNA methylation, offer promise in early cancer detection. The present study compared commercial kits for analyzing DNA from cervical liquid cytology samples in cancer screening. Rapid bisulfite conversion kits using silica spin-columns and magnetic beads were assessed against standard DNA extraction and bisulfite conversion methods for profiling DNA methylation using quantitative methylation-specific PCR. ß-actin amplification indicated the suitability of small sample volumes for methylation studies using either the pellet or supernatant (cell-free DNA) parts. Comparison of Bisulfite Conversion Kit-Whole Cell (Abcam), Methylamp Bisulfite Modification (Epigentek), EpiTect Fast LyseAll Bisulfite Kit (Qiagen GmbH) and EZ DNA Methylation-Direct Kit (Zymo Research Corp.) showed no significant differences in ß-actin cycle threshold values. EZ-96 DNA Methylation-Lightning MagPrep (Zymo Research Corp.), a hybrid kit in a 96-well plate format, exhibited swift turnaround time and similar amplification efficiency. Automation with magnetic bead kits increased throughput without compromising amplification efficiency in open PCR systems. Cost analysis favored direct kits over the gold standard manual protocol. This comparison aids in selecting cost-effective DNA methylation diagnostic tests. The present study confirmed comparable kit performance in methylation-based analysis, highlighting the adequacy of cytology samples and the potential of bodily fluids as alternatives for liquid biopsy.

Identification of a methylation panel as an alternative triage to detect CIN3+ in hrHPV-positive self-samples from the population-based cervical cancer screening programme.

BACKGROUND: The Dutch population-based cervical cancer screening programme (PBS) consists of primary high-risk human papilloma virus (hrHPV) testing with cytology as triage test. In addition to cervical scraping by a general practitioner (GP), women are offered self-sampling to increase participation. Because cytological examination on self-sampled material is not feasible, collection of cervical samples from hrHPV-positive women by a GP is required. This study aims to design a methylation marker panel to detect CIN3 or worse (CIN3+) in hrHPV-positive self-samples from the Dutch PBS as an alternative triage test for cytology. METHODS: Fifteen individual host DNA methylation markers with high sensitivity and specificity for CIN3+ were selected from literature and analysed using quantitative methylation-specific PCR (QMSP) on DNA from hrHPV-positive self-samples from 208 women with CIN2 or less (< CIN2) and 96 women with CIN3+. Diagnostic performance was determined by area under the curve (AUC) of receiver operating characteristic (ROC) analysis. Self-samples were divided into a train and test set. Hierarchical clustering analysis to identify input methylation markers, followed by model-based recursive partitioning and robustness analysis to construct a predictive model, was applied to design the best marker panel. RESULTS: QMSP analysis of the 15 individual methylation markers showed discriminative DNA methylation levels between < CIN2 and CIN3+ for all markers (p < 0.05). The diagnostic performance analysis for CIN3+ showed an AUC of ≥ 0.7 (p < 0.001) for nine markers. Hierarchical clustering analysis resulted in seven clusters with methylation markers with similar methylation patterns (Spearman correlation> 0.5). Decision tree modeling revealed the best and most robust panel to contain ANKRD18CP, LHX8 and EPB41L3 with an AUC of 0.83 in the training set and 0.84 in the test set. Sensitivity to detect CIN3+ was 82% in the training set and 84% in the test set, with a specificity of 74% and 71%, respectively. Furthermore, all cancer cases (n = 5) were identified. CONCLUSION: The combination of ANKRD18CP, LHX8 and EPB41L3 revealed good diagnostic performance in real-life self-sampled material. This panel shows clinical applicability to replace cytology in women using self-sampling in the Dutch PBS programme and avoids the extra GP visit after a hrHPV-positive self-sampling test.

IL10 hypomethylation is associated with the risk of gastric cancer.

Interleukin-10 (IL10), a pleiotropic cytokine secreted by type-2 helper (Th2) T cells, contributes to the oncogenic activation or inactivation of tumor-suppressor genes. The present study investigated whether hypomethylation of IL10 CpG island (CGI) was associated with the risk of developing gastric cancer (GC) and the prognosis of patients with GC. A fragment (hg18, chr1: 206945638-206945774) at the CGI of IL10 was selected for the present methylation assay. Quantitative methylation-specific PCR was used to evaluate the methylation of IL10 CGI in 117 tumor samples from patients with GC. The results demonstrated that IL10 CGI methylation was significantly lower in the tumor tissues compared with that in the paired adjacent non-tumor tissues (median percentage of methylated reference, 29.16 vs. 42.82%, respectively; P=4×10-8). Furthermore, results from receiver operating characteristic curve analysis identified a significant area under the curve of 0.706, with a sensitivity and a specificity of 77.8 and 58.1%, respectively, between cancer tissues and paired adjacent non-tumor tissues. Furthermore, the methylation of IL10 CGI was significantly associated with patients' age at diagnosis (r=-0.201; P=0.03). Subgroup analyses demonstrated that the association between IL10 CGI hypomethylation and the risk of GC was specific for patients with low differentiation (P=1×10-7) and Borrmann types III+IV (P=1×10-7). In addition, IL10 CGI hypomethylation was significantly associated with the risk of GC for patients without smoking history (P=3×10-7) or a family history of cancer (P=2×10-7). The results from Kaplan-Meier survival analysis demonstrated that IL10 CGI hypomethylation was associated with a significantly shorter overall survival of patients with GC (P=0.041). Similar results were identified for patients with GC who did not have smoking history (P=0.037) or a family history of cancer (P=0.049). The results from this study demonstrated that IL10 CGI hypomethylation may be considered as a potential biomarker for the diagnosis and prognosis of patients with GC in the Chinese population.

Development of Quantitative Methylation-Specific Droplet Digital PCR (ddMSP) for Assessment of Natural Tregs.

Regulatory T cells (Tregs) suppress immune responses in vivo in an antigen-specific manner. Of clinical relevance, Tregs can be isolated and expanded in vitro while maintaining immunoregulatory function. Tregs are classified as CD4+CD25highCD127low FOXP3+ cells. Demethylation of the Treg-specific demethylation region (TSDR) of FOXP3 is found in natural Tregs (nTregs). We report a method for the characterization of the differential methylation pattern of the FOXP3 TSDR in patient-derived and expanded nTregs. Human TSDR sequences from nTregs (unmethylated sequence) and pancreatic (methylated sequence) cells were amplified and cloned into plasmids. A droplet digital TaqMan probe-based qPCR (ddPCR) assay using methylation-specific primers and probes was employed to quantify unmethylated and methylated sequences. The methylation-specific droplet digital PCR (ddMSP) assay was specific and selective for unmethylated DNA in mixtures with methylated DNA in the range of 5000 copies/µL to less than 1 copy/µL (R 2 = 0.99) even in the presence of non-selective gDNAs. CD4+CD25highCD127lowFOXP3+ human nTregs, in the presence of Dynabeads or activators, were expanded for 21 days. There was a decrease in the unmethylated ratio of Tregs after expansion with essentially the same ratio at days 10, 14, and 17. However, the activator expanded group showed a significant decrease in unmethylated targets at day 21. The suppression activity of activator-expanded nTregs at day 21 was decreased compared to cells expanded with Dynabeads. These data suggest that the ddMSP can quantitatively monitor nTreg expansion in vitro. These data also indicate that the assay is sensitive and specific at differentiating nTregs from other cells and may be useful for rapid screening of nTregs in clinical protocols.

Hypermethylated Promoters of Secreted Frizzled-Related Protein Genes are Associated with Colorectal Cancer.

Colorectal cancer (CRC) is one of the leading causes of death worldwide. Aberrant DNA methylation has been recognized as one of the most common molecular alterations in CRC. The goal of this study was to investigate the diagnostic value of SFRP1 and SFRP2 methylation for CRC. A total of 80 pairs of CRC patients were recruited to test the association of SFRP1 and SFRP2 promotor methylation with CRC. Methylation assay was performed using quantitative methylation-specific polymerase chain reaction (qMSP) method. In this study, we found the methylation levels of SFRP1 and SFRP2 in CRC tumor tissues were significantly higher than those in the adjacent non-tumor tissues (SFRP1: P = 2E-5; SFRP2: P = 0.014). Further bioinformatics analysis of TCGA data confirmed the association of the two genes with CRC (SFRP1: P = 7E-21; SFRP2: P = 5E-24). Luciferase reporter gene assay showed that the recombinant plasmids with SFRP1 and SFRP2 fragments could significantly enhance promoter activity (SFRP1: P = 0.002; SFRP2: P = 0.004). In addition, SFRP1 and SFRP2 methylation were inversely correlated with the mRNA expression displayed by TCGA data mining (SFRP1: r = -0.432, P = 4E-11; SFRP2: r = -0.478, P = 1E-13). GEO data analysis indicated that SFRP1 and SFRP2 expression were increased in three CRC cell lines (COLO320, HCT116 and HT29) after 5'-AZA-deoxycytidine treatment, suggesting that DNA methylation played an important role in regulating gene expression of the two genes. Our results confirmed that promoter methylation of SFRP1 and SFRP2 contributed to the risk of CRC.

Quantitative Methylation-Specific PCR: A Simple Method for Studying Epigenetic Modifications of Cell-Free DNA.

Aberrant DNA methylation of cell-free circulating DNA (cfDNA) has recently gained attention for its use as biomarker in cancer diagnosis, prognosis, and prediction of therapeutic response. Quantification of cfDNA methylation levels requires methods with high sensitivity and specificity due to low amounts of cfDNA available in plasma, high degradation of cfDNA, and/or contamination with genomic DNA. To date, several approaches for measuring cfDNA methylation have been established, including quantitative methylation-specific PCR (qMSP), which represents a simple, fast, and cost-effective technique that can be easily implemented into clinical practice. In this chapter, we provide a detailed protocol for SYBR Green qMSP analysis which is currently used in our laboratory for cfDNA methylation detection. Useful information regarding successful qMSP primers design are also provided.

Genes Located on 18q23 Are Epigenetic Markers and Have Prognostic Significance for Patients with Head and Neck Cancer.

Loss of heterozygosity (LOH) on chromosome 18q23 is associated with significantly decreased survival in head and neck cancer. In agreement with such tumor suppressive roles, the loss of function of genes located in this region can be achieved through LOH and promotor hypermethylation. In this study, the methylation status of promoters of 18q23 genes in 243 head and neck cancer patients was assessed by quantitative methylation-specific PCR. Promoter methylation was then compared to various clinical characteristics and patient survival. GALR1 and SALL3 promoter methylation correlated with reduced disease-free survival (log-rank test, p = 0.018 and p = 0.013, respectively). Furthermore, based on multivariate Cox proportional hazards analysis, these methylation events were associated with poor disease-free survival, with hazard ratios of 1.600 (95% confidence interval: CI, 1.027⁻2.493; p = 0.038) and 1.911 (95% CI, 1.155⁻3.162; p = 0.012), respectively. By comparison, GALR1 and SALL3 methylation were not prognostic for overall survival in The Cancer Genome Atlas (TCGA) cohort. Our findings suggest that the methylation status of 18q23 genes could serve as important biomarkers for the prediction of clinical outcomes in well-annotated head and neck squamous cell carcinoma cohorts. GALR1 and SALL3 methylation could thus help to facilitate risk stratification for individualized treatment.

Assays for Determining Repeat Number, Methylation Status, and AGG Interruptions in the Fragile X-Related Disorders.

Knowledge of the CGG•CCG-repeat number, AGG interruption status, and the extent of DNA methylation of the FMR1 gene are vital for both diagnosis of the fragile X-related disorders and for basic research into disease mechanisms. We describe here assays that we use in our laboratory to assess these parameters. Our assays are PCR-based and include one for repeat size that can also be used to assess the extent of methylation and a related assay that allows the AGG interruption pattern to be reliably determined even in women. A second more quantitative methylation assay is also described. We also describe our method for cloning of repeats to generate the reference standards necessary for the accurate determination of repeat number and AGG interruption status.

DNA Methylation Analysis.

DNA methylation is a process by which methyl groups are added to cytosine or adenine. DNA methylation can change the activity of the DNA molecule without changing the sequence. Methylation of 5-methylcytosine (5mC) is widespread in both eukaryotes and prokaryotes, and it is a very important epigenetic modification event, which can regulate gene activity and influence a number of key processes such as genomic imprinting, cell differentiation, transcriptional regulation, and chromatin remodeling. Profiling DNA methylation across the genome is critical to understanding the influence of methylation in normal biology and diseases including cancer. Recent discoveries of 5-methylcytosine (5mC) oxidation derivatives including 5-hydroxymethylcytosine (5hmC), 5-formylcytsine (5fC), and 5-carboxycytosine (5caC) in mammalian genome further expand our understanding of the methylation regulation. Genome-wide analyses such as microarrays and next-generation sequencing technologies have been used to assess large fractions of the methylome. A number of different quantitative approaches have also been established to map the DNA epigenomes with single-base resolution, as represented by the bisulfite-based methods, such as classical bisulfite sequencing, pyrosequencing etc. These methods have been used to generate base-resolution maps of 5mC and its oxidation derivatives in genomic samples. The focus of this chapter is to provide the methodologies that have been developed to detect the cytosine derivatives in the genomic DNA.

CDH23 Methylation Status and Presbycusis Risk in Elderly Women.

Introduction: Presbycusis, an age-related hearing impairment (ARHI) disease, is the most common cause for HI in adults worldwide. One of the best candidate genes for ARHI susceptibility is Cadherin 23 (CDH23) which encodes stereocilia tip-links of the inner ear sensory hair cell. Although alterations in the methylation status of CpG dinucleotides across various genes were reported to be associated with HI, methylation changes in CDH23 gene have not been reported previously. Objectives: This study aimed at investigating whether DNA methylation level of CDH23 gene at intragenic CpG island overlapping an exonic-intronic region at position chr10:73565570-73565827 (GRCh37/hg19) could be risk factor associated with ARHI. Materials and Methods: We screened for methylation changes in this particular position for CDH23 gene in 50 blood samples of elderly women affected with presbycusis and healthy control cohort. Methylation of CpG sites were assessed using Quantitative methylation-specific PCR (qMSP) following sodium bisulfite DNA conversion chemistry. Methylation levels were normalized against TSH2B reference gene. Results: DNA methylation analysis for the common CpG islands in CDH23 gene revealed 3.27-folds significant increase (p < 0.0001) in methylation profile for ARHI women as compared to healthy controls with an elevated risk odds ratio (OR) of 2.219 [95% CI 1.071-4.597]. Conclusion: Our study is the first of its kind to prove that higher CpG site methylation levels in CDH23 gene are likely to be associated with ARHI.

Host-cell DNA methylation patterns during high-risk HPV-induced carcinogenesis reveal a heterogeneous nature of cervical pre-cancer.

Cervical cancer development following a persistent infection with high-risk human papillomavirus (hrHPV) is driven by additional host-cell changes, such as altered DNA methylation. In previous studies, we have identified 12 methylated host genes associated with cervical cancer and pre-cancer (CIN2/3). This study systematically analyzed the onset and DNA methylation pattern of these genes during hrHPV-induced carcinogenesis using a longitudinal in vitro model of hrHPV-transformed cell lines (n = 14) and hrHPV-positive cervical scrapings (n = 113) covering various stages of cervical carcinogenesis. DNA methylation analysis was performed by quantitative methylation-specific PCR (qMSP) and relative qMSP values were used to analyze the data. The majority of genes displayed a comparable DNA methylation pattern in both cell lines and clinical specimens. DNA methylation onset occurred at early or late immortal passage, and DNA methylation levels gradually increased towards tumorigenic cells. Subsequently, we defined a so-called cancer-like methylation-high pattern based on the DNA methylation levels observed in cervical scrapings from women with cervical cancer. This cancer-like methylation-high pattern was observed in 72% (38/53) of CIN3 and 55% (11/20) of CIN2, whereas it was virtually absent in hrHPV-positive controls (1/26). In conclusion, hrHPV-induced carcinogenesis is characterized by early onset of DNA methylation, typically occurring at the pre-tumorigenic stage and with highest DNA methylation levels at the cancer stage. Host-cell DNA methylation patterns in cervical scrapings from women with CIN2 and CIN3 are heterogeneous, with a subset displaying a cancer-like methylation-high pattern, suggestive for a higher cancer risk.

The more potential performance of nidogen 2 methylation by tissue or plasma DNA over brichoalveolar lavage DNA in diagnosis of nonsmall cell lung cancer.

BACKGROUND: Nonsmall cell lung cancer (NSCLC) is one of the leading incidence and mortality of malignant tumors worldwide. While aberrant DNA methylation is a frequent event occurred during NSCLC carcinogenesis and development, therefore holding the potential to predict the process of tumor development. This study aims to explore the feasibility of gene nidogen 2 (NID2) as the diagnostic biomarker for NSCLC. MATERIALS AND METHODS: Quantitative methylation specific polymerase chain reaction of NID2 has been done among the following sample panels: For tissue methylation evaluation, we collected 96 cases of NSCLC versus 18 cases of noncancerous lung lesions (NCLLs); 46 from the 96 NSCLC patients also provided DNA of bronchoalveolar lavage (BAL) and plasma sample, the methylation status of which are assessed against 12 cases of NCLL for BAL and 30 cases of NCLL for plasma samples, respectively. RESULTS: The methylation rate of NID2 in NSCLC versus NCLL is evaluated as: In tissue 59.40% versus 16.67%, (P = 0.0001); in BAL 30.43% versus 16.67% (P = 0.1640); in plasma 45.65% versus 20.00% (P = 0.0191). CONCLUSIONS: Our study revealed the frequent occurrence of aberrant NID2 methylation in NSCLC and peripheral blood, which might be useful as a biomarker to predict NSCLC or to screen the high-risk population for NSCLC.