Percentage Of Methylated Reference Research Articles

Targeted demethylation of the CDO1 promoter based on CRISPR system inhibits the malignant potential of breast cancer cells.

Cysteine dioxygenase 1 (CDO1) is frequently methylated, and its expression is decreased in many human cancers including breast cancer (BC). However, the functional and mechanistic aspects of CDO1 inactivation in BC are poorly understood, and the diagnostic significance of serum CDO1 methylation remains unclear. We performed bioinformatics analysis of publicly available databases and employed MassARRAY EpiTYPER methylation sequencing technology to identify differentially methylated sites in the CDO1 promoter of BC tissues compared to normal adjacent tissues (NATs). Subsequently, we developed a MethyLight assay using specific primers and probes for these CpG sites to detect the percentage of methylated reference (PMR) of the CDO1 promoter. Furthermore, both LentiCRISPR/dCas9-Tet1CD-based CDO1-targeted demethylation system and CDO1 overexpression strategy were utilized to detect the function and underlying mechanism of CDO1 in BC. Finally, the early diagnostic value of CDO1 as a methylation biomarker in BC serum was evaluated. CDO1 promoter was hypermethylated in BC tissues, which was related to poor prognosis (p<.05). The CRISPR/dCas9-based targeted demethylation system significantly reduced the PMR of CDO1 promotor and increased CDO1 expression in BC cells. Consequently, this leads to suppression of cell proliferation, migration and invasion. Additionally, we found that CDO1 exerted a tumour suppressor effect by inhibiting the cell cycle, promoting cell apoptosis and ferroptosis. Furthermore, we employed the MethyLight to detect CDO1 PMR in BC serum, and we discovered that serum CDO1 methylation was an effective non-invasive biomarker for early diagnosis of BC. CDO1 is hypermethylated and acts as a tumour suppressor gene in BC. Epigenetic editing of abnormal CDO1 methylation could have a crucial role in the clinical treatment and prognosis of BC. Additionally, serum CDO1 methylation holds promise as a valuable biomarker for the early diagnosis and management of BC.

Plasma central carbon metabolite changes associated with KRAS mutation and circulating tumor DNA (ctDNA) status in colorectal cancer (CRC).

181 Background: KRAS mutations have been widely characterized as markers of poor prognosis in CRC. In stage IV CRC, KRAS mutations are predictive of benefit to anti-EGFR therapy. ctDNA has increasingly been recognized as a prognostic biomarker in CRC as well. We evaluated the association between plasma metabolites and KRAS mutation or ctDNA status in a longitudinal, observational cohort of patients with stage I-IV CRC. Methods: This was a retrospective analysis of prospectively collected blood samples from a single-institute cohort of patients with stage I-IV CRC. All blood samples were collected at pre-chemotherapy baseline. A modified Epi proColon 2.0 CE (Epigenomics AG) assay was used for plasma ctDNA testing on the methylated SEPTIN9 gene (mSEPT9). ctDNA positivity was defined as a mSEPT9 percentage of methylation reference (PMR) value greater than zero. Up to 150 metabolites of central carbon metabolism were analyzed by mass spectrometry and high-performance liquid chromatography. Analytes were compared by relative area under the curve (AUC) and differences evaluated by ANOVA. The mean AUC was used in patients with metabolites measured from &gt; 1 timepoint of collection. Patients were stratified by ctDNA status (positive or negative) and KRAS mutant (MT) or wildtype (WT) status. Results: A total of 32 patients were included with median age 65 years (range 20-90). The majority were female (53%) and had stage IV disease (78%). Of 25 patients with stage IV CRC, 88% had pre-chemotherapy blood samples collected in the first-line setting. Most patients were KRAS MT (44%) compared to KRAS WT (37%) or unknown KRAS status (19%). The most common KRAS MT subtypes were G12D (29%), G12V (29%), G13D (21%), and G12C (14%). The mean overall survival in this cohort was 27.4 months while the mean mSEPT9 PMR value was 2553.6. When stratified by ctDNA status, ctDNA positivity was associated with decreased levels of essential amino acids (lysine, methionine, threonine) and the non-essential amino acid arginine (all p &lt; 0.05). Compared to KRAS WT tumors, KRAS MT tumors were associated with increased levels of proline, phenylalanine, and intermediates of glycolysis (lactate), MTA cycle (SAM, 5-Methioadenosine), and O-GlcNAcylation (GlcNac, all p &lt; 0.05). Conclusions: We are the first to demonstrate the feasibility of associating central carbon metabolites with ctDNA and KRAS mutation status. As ctDNA positivity and KRAS MT status have evolving prognostic potential in CRC, associated metabolic signatures may identify metabolic pathways for novel biomarker development. Our findings also show that KRAS MT CRC appears to be enriched in intermediates of glycolytic, methyl donor, and O-GlcNAcylation pathways.

Racial Disparities in Methylation of NRF1, FTO, and LEPR Gene in Childhood Obesity

Childhood obesity has affected the health of millions of children around the world despite vigorous efforts by health experts. The obesity epidemic in the United States has disproportionately afflicted certain racial and ethnic minority groups. African American children are more likely than other children to have obesity-related risk factors such as hyperlipidemia, diabetes, cardiovascular disease, and coronavirus disease (COVID-19). For the reduction in obesity-related health inequalities to be successful, it is essential to identify the variables affecting various groups. A notable advancement in epigenetic biology has been made over the past decade. Epigenetic changes like DNA methylation impact on many genes associated with obesity. Here, we evaluated the DNA methylation levels of the genes NRF1, FTO, and LEPR from the saliva of children using real-time quantitative PCR-based multiplex MethyLight technology. ALU was used as a reference gene, and the Percent of Methylated Reference (PMR) was calculated for each sample. European American children showed a significant increase in PMR of NRF1 and FTO in overweight/obese participants compared to normal weight, but not in African American children. After adjusting for maternal education and annual family income by regression analysis, the PMR of NRF1 and FTO was significantly associated with BMI z-score only in European American children. While for the gene LEPR, African American children had higher methylation in normal weight participants as compared to overweight/obese and no methylation difference in European American children. The PMR of LEPR was significantly negative associated with the obesity measures only in African American children. These findings contribute to a race-specific link between NRF1, FTO, and LEPR gene methylation and childhood obesity.

DNA methylation and copy number variation of the complement C4A gene in schizophrenia

The complement gene C4A has been implicated in schizophrenia through genome-wide association studies (GWAS), in vitro and animal studies. However, few studies have examined epigenetic modification of the C4A gene in schizophrenia. We hypothesized that DNA methylation levels of the C4A gene are altered in schizophrenia, and the interaction between DNA methylation and the copy number (CN) of the C4A gene leads to the higher C4 levels seen in schizophrenia. In this study, C4A DNA methylation levels, C4A CN and C4 levels were measured in the peripheral blood of 183 schizophrenia cases (SZ) and 212 healthy controls (HC). The Positive and Negative Syndrome Scale (PANSS) was used to determine the severity of psychopathology in the cases. MethyLight assay was used to measure DNA methylation levels and droplet digital polymerase chain reaction (ddPCR) was used for the calculated copy number (cCN) determination. The cCNs were also grouped into copy number variation (CNV) types: deletion, normal, and duplication. Methylation levels were measured as percentage of methylated reference (PMR) values. Both C4A PMR (rs = 0.469, p < 0.001) and C4A cCN (rs = 0.210, p < 0.001) were positively correlated with C4 levels, which in turn were higher in SZ than HC. However, we found no difference in C4A PMR values or C4A CNV types between SZ and HC. Our results suggest the potential involvement of an epigenetic modification and CNV of C4A in the upregulation of complement C4 in schizophrenia.

Quantitative detection of SRY-Box 21 (SOX21) gene promoter methylation as a stool-based noninvasive biomarker for early diagnosis of colorectal cancer by MethyLight method.

In recent years, the study of potential epigenetic biomarkers in feces has been an attractive research approach for the noninvasive diagnosis of colorectal cancer (CRC). The aim of this study was to evaluate the stool-based DNA methylation potential of SRY-Box 21 (SOX21) gene promoter as an appropriate candidate biomarker for differentiating CRC patients and healthy individuals for the first time. The MethyLight method was performed to analyze the methylation status of SOX21 gene promoter in fecal samples from 40 patients with CRC and 40 healthy controls. In addition, the diagnostic efficiency of measuring the hypermethylated SOX21 gene in the feces to the fecal occult blood test (FOBT) was compared. The percentage of methylated reference (PMR) values in the stool of CRC patients (median 1.44) was higher than those of healthy individuals (median 0.00) (P < 0.001). A sensitivity of 72.5% and specificity of 100% were obtained for SOX21 gene promoter methylation status and 29 of the patients were considered as positive in methylation status. There was no significant association between PMR values and demographic/clinicopathological features (P > 0.05). The results of the present study demonstrated that the stool-based assay of SOX21 gene promoter methylation has a relatively high sensitivity and specificity and it may serve as a noninvasive biomarker for early detection of CRC. However, more studies with a wide range of samples are required to further confirm the role of hypermethylation of SOX21 in the early CRC diagnosis.

The values of AHCY and CBS promoter methylation on the diagnosis of cerebral infarction in Chinese Han population

BackgroundThe goal of our study is to investigate whether the methylation levels of AHCY and CBS promoters are related to the risk of cerebral infarction by detecting the methylation level of AHCY and CBS genes.MethodsWe extracted peripheral venous blood from 152 patients with cerebral infarction and 152 gender- and age-matched healthy controls, and determined methylation levels of AHCY and CBS promoters using quantitative methylation-specific polymerase chain reaction. We used the percentage of methylation reference (PMR) to indicate gene methylation level.ResultsWe compared the promoter methylation levels of two genes (AHCY and CBS) in peripheral blood DNA between the cerebral infarction case group and the control group. Our study showed no significant difference in AHCY promoter methylation between case and control. Subgroup analysis by gender showed that the methylation level of AHCY in males in the case group was lower than that in the control group, but the difference was not statistically significant in females. In a subgroup analysis by age, there was no significant difference in the AHCY methylation level between the case and control in the young group (≤44 years old). However, the level of AHCY gene methylation in the middle-aged group (45–59 years old) was significantly higher and the aged group (≥60 years old) was significantly lower than that in the control groups. However, CBS promoter methylation levels were significantly lower in the case group than in the control group (median PMR: 70.20% vs 104.10%, P = 3.71E-10). In addition, the CBS methylation levels of males and females in the case group were significantly lower than those in the control group (male: 64.33% vs 105%, P = 2.667E-08; female: 78.05% vs 102.8%, P = 0.003). We also found that the CBS levels in the young (23–44), middle-aged (45–59), and older (60–90) groups were significantly lower than those in the control group (young group: 69.97% vs 114.71%; P = 0.015; middle-aged group: 56.04% vs 91.71%; P = 6.744E-06; older group: 81.6% vs 119.35%; P = 2.644E-04). Our ROC curve analysis of CBS hypomethylation showed an area under the curve of 0.713, a sensitivity of 67.4%, and a specificity of 74.0%.ConclusionOur study suggests that hypomethylation of the CBS promoter may be closely related to the risk of cerebral infarction and may be used as a non-invasive diagnostic biomarker for cerebral infarction.

FANCF hypomethylation is associated with colorectal cancer in Han Chinese.

Fanconi anemia complement group F (FANCF) is known to be involved in DNA repair, and the overexpression of FANCF protein leads to cell proliferation and ultimately to cancer. The purpose of this study was to assess whether FANCF methylation was associated with colorectal cancer (CRC). A case-control experiment was conducted to study the association between FANCF methylation and CRC. We used quantitative methylation-specific PCR to measure the FANCF promoter methylation, and the percentage of methylation reference (PMR) to quantify the FANCF promoter methylation level. To investigate the effect of the selected FANCF fragment on gene expression regulation, we also performed a dual-luciferase reporter gene assay. The results indicated that FANCF methylation in CRC tumor tissues was significantly lower than that in the nontumor tissues (median PMR: 44.86% vs. 65.77%, p=0.00001). Analysis of receiver-operating characteristic curves showed that FANCF hypomethylation had a diagnostic value for CRC (area under curve [AUC]: 0.670, sensitivity: 55.8%, specificity: 71.7%, p=0.00001). The dual-luciferase reporter assay showed that the FANCF fragment upregulated gene expression (fold change: 1.93, p=0.002). Research demonstrates for the first time that FANCF hypomethylation is significantly associated with CRC risk. FANCF hypomethylation may ultimately increase the risk of CRC by upregulating the expression of FANCF.

LEPR hypomethylation is significantly associated with gastric cancer in males

BackgroundPrevious study has shown LEPR is a candidate gene of prediction and treatment of gastric cancer (GC). The purpose of this study was to test whether LEPR methylation could predict the risk of GC. Materials and methodsTumor tissues and 5-cm adjacent non-tumor tissues from 117 newly diagnosed and untreated GC patients were collected for the current methylation study. LEPR methylation levels were determined by quantitative methylation specific PCR (qMSP), and the methylation level of LEPR was described by the percentage of methylated reference (PMR). ResultsOur results showed that LEPR methylation levels were significantly lower in tumor tissues than those in adjacent non-tumor tissues (median PMR: 36.64% vs. 50.29%, P = 1E-4). In addition, LEPR methylation levels were found to be significantly associated with platelet (r = −0.198, P = .037). Further subgroup analysis showed that the association of LEPR promoter hypomethylation with GC was specific to males (males: P = 7E-5; females: P = .500). Notably, significant hypomethylation of LEPR promoter was found only in GC patients without recurrence (P = .002) but not in GC patients with recurrence (P = .146). The AUC of LEPR hypomethylation for identification of GC risk was 0.649 with a sensitivity of 67.5% and a specificity of 63.2%. In addition, the AUC of LEPR hypomethylation in males was 0.685 with a sensitivity of 68.4% and a specificity of 69.6%. ConclusionLEPR hypomethylation can be used to predict the risk of GC in males. And it might also have the potential to predict the recurrence in GC patients.

Hypermethylation of dihydrofolate reductase promoter increases the risk of hypertension in Chinese.

Background:DNA methylation was considered to play an important role in hypertension. However, the direct association between dihydrofolate reductase (DHFR) promoter methylation and hypertension remains unclear. We thus aimed to investigate the relationship between DNA methylation of DHFR promoter and hypertension.Materials and Methods:A total of 371 hypertensive patients (diastolic blood pressure ≥90 mmHg and/or systolic blood pressure ≥140 mmHg or a history of antihypertensive treatment) and 320 age- and sex-matched healthy controls from the Hypertension Management Information System in Nanshan Community Health Service Centers were included in this case–control study. Quantitative methylation-specific polymerase chain reaction was used to measure the level of DHFR promoter methylation, which was presented as the percentage of methylated reference (PMR). A multivariate logistic regression model was used to explore the risk of DHFR promoter methylation.Results:Our results indicated that the level of DHFR promoter methylation was higher in hypertensive patients (median PMR, 34.32%; interquartile range, 11.34–119.60) than in healthy controls (median PMR, 18.45%; interquartile range, 8.16–35.40) (P < 0.001). Multivariable analysis showed that the risk of DHFR promoter hypermethylation was significantly higher in hypertensive patients than in healthy controls (odds ratio = 3.94, 95% confidence interval = 2.56–6.02, P < 0.001). Furthermore, hypermethylation was positively associated with sex, high blood homocysteine levels, and alcohol drinking. In particular, the area under the receiver operating characteristic curve was 0.688 (0.585–0.668) for the male hypertensive patients, suggesting the potential diagnostic value of DHFR promoter methylation in male hypertension.Conclusion:Our results demonstrated that DHFR promoter hypermethylation is positively associated with the risk of hypertension in Chinese.

Association of human serotonin receptor 4 promoter methylation with autism spectrum disorder.

Human serotonin receptor 4 (HTR4) encodes a 5-HT4 receptor involved in learning, memory, depression, anxiety, and feeding behavior. The aim of this study was to investigate the association between the deoxyribonucleic acid (DNA) methylation of HTR4 promoter and autism spectrum disorder (ASD), a disease characterized by communication disorder and repetitive or restrictive behavior.Peripheral blood DNA was obtained from 61 ASD children and 66 healthy children, and the DNA methylation of HTR4 promoter was assessed by quantitative methylation-specific polymerase chain reaction. We used percentage of methylated reference (PMR) to represent DNA methylation level.Due to significant age differences between ASD cases and controls (3 [2, 5] years and 6 [5, 6] years, P = 3.34E-10), we used binary logistic regression analysis for adjustment. Our results showed that the DNA methylation levels of HTR4 promoter were significantly lower in children with ASD than in healthy children (median PMR: 66.23% vs 94.31%,P = .028, age-adjusted P = .034). In addition, the DNA methylation of HTR4 promoter was inversely associated with age in male ASD cases (total cases: r = –0.283, P = .027; male cases: r = –0.431, P = .002; female cases: r = –0.108, P = .752). Dual-luciferase reporter gene assay showed that the reporter gene expression in the strain with recombinant pGL3-promoter-HTR4 plasmid was significantly higher than that in the strain with pGL3-promoter plasmid (fold change = 2.01, P = .0065), indicating that the HTR4 promoter fragment may contain transcription factors to upregulate promoter activity.Our study suggested that hypomethylation of the HTR4 promoter is a potential biomarker for predicting the risk of male ASD.

A male-specific association between AGTR1 hypermethylation and coronary heart disease.

The AGTR1 gene encodes angiotensin II receptor type 1, which is involved in cardiovascular diseases such as coronary heart disease (CHD). In the current study, we analyzed AGTR1 promoter methylation level in a Han Chinese population by SYBR green-based quantitative methylation-specific PCR (qMSP). We collected blood samples from 761 CHD patients and 398 non-CHD controls at the Ningbo First Hospital. A data mining analysis was also performed to explore the association between AGTR1 methylation and AGTR1 gene expression, using datasets from the cBioPortal for Cancer Genomics and the Gene Expression Omnibus (GEO) database. Our results showed a significantly higher percentage of methylated reference (PMR) of AGTR1 in male CHD patients compared with male non-CHD controls (median PMR: 2.12% vs. 0.59%, p = 0.037). The data mining analysis showed that AGTR1 expression was significantly increased in human hepatoma HepG2 cells treated with the demethylation agent 5-aza-2’-deoxycytidine (fold = 3.12, p = 0.009). Further data mining analysis using the cholangiocarcinoma (TCGA, PanCancer Atlas) data indicated an inverse association between AGTR1 methylation and AGTR1 expression (r = -0.595, p = 1.29E-04). Overall, our results suggest that AGTR1 methylation is involved in the regulation of AGTR1 gene expression and that AGTR1 hypermethylation is associated with CHD in males. These findings may provide new clues about the pathogenesis of CHD.

Gout in males: a possible role for COMT hypomethylation

Gout is a common inflammatory disease, and the prevalence of gout in men is significantly higher than in women. Catechol-O-methyltransferase (COMT) regulates dopamine activity and metabolism, thereby participating in the uric acid metabolism, which in turn affects the occurrence of gout. Our study aimed to investigate the association between COMT methylation and gout in men. This study involved 57 male gout patients and 103 age-matched healthy men. We used quantitative methylation-specific polymerase chain reaction (qMSP) to determine DNA methylation levels in the blood. The COMT methylation level was represented by the percentage of methylation reference (PMR). Our results showed that COMT methylation levels were significantly lower in gout patients than in the control group (median PMR 9.50 vs 31.34, p = 3E-5). The area under the curve (AUC) was 0.701 (95% CI 0.611-0.790, p = 2.7E-5) with a sensitivity of 68% and a specificity of 68.4%. Our study found that there was a significant correlation between COMT hypomethylation and the risk of gout in males, and this provides an epigenetic mechanism of COMT in gout. COMT hypomethylation might be used as a potential diagnostic biomarker for gout in the future.

Significant association of EED promoter hypomethylation with colorectal cancer.

Colorectal cancer (CRC) is one of the most common and serious types of malignancy worldwide. The embryonic ectoderm development (EED) gene is important to maintain transcriptional repressive states of genes over successive cell generations. The present study aimed to investigate the association between EED methylation and CRC. A total of 111 CRC tissue samples, 111 paired para-tumor tissues and 20 colorectal normal tissues were obtained for EED methylation assay, which was performed using a quantitative methylation-specific polymerase chain reaction. The percentage of methylated reference was calculated to represent the DNA methylation level. A dual-luciferase reporter gene assay was used to detect the gene promoter activity of a EED fragment. The current results revealed a significant difference in the EED methylation levels among tumor, para-tumor and normal colorectal tissues (tumor vs. para-tumor vs. normal, 5.03±4.61 vs. 8.65±11.50 vs. 40.12±45.31; F=45.014; P<0.0001). The dual-luciferase reporter gene assay demonstrated that the transcriptional activity of recombinant pGL3-EED plasmid was significantly higher compared with that of the pGL3-Basic control vector (fold-change, 3.15; P=0.014), which suggests the EED fragment can promote gene expression. In conclusion, the present study demonstrated that EED hypomethylation may be an important factor associated with CRC.

Association of HOXA9 Promoter Hypomethylation With Colorectal Cancer

Background: The HOXA9 gene encodes a DNA-binding transcription factor that is closely related to cancer, including colorectal cancer (CRC). In this study, we first explored whether HOXA9 promoter methylation was associated with CRC. Materials and Methods: Quantitative methylation-specific PCR (qMSP) was used to detect HOXA9 methylation levels in 76 patients with tumor tissues and paired adjacent non-tumor tissues. The percentage of methylation reference (PMR) was used to indicate the level of gene methylation. Results: Our results showed that the methylation level of HOXA9 in tumor tissues was significantly lower than that of matched adjacent non-tumor tissues (median PMR, 4.61% vs 10.04%, P = 0.003). The similar significant difference was also found in 45 pairs of CRC samples from the Cancer Genome Atlas (TCGA) database (cg21942490, P = 0.039; cg22055728, P = 0.001). In addition, TCGA data analysis also showed a significant inverse correlation between HOXA9 methylation level and its gene expression (r = -0.438, P = 3.71E-59). Data analysis from Gene Expression Omnibus (GEO) showed that cells treated with demethylating reagent significantly up-regulated HOXA9 expression (fold change > 1.76, P = 0.009). Conclusion: Our results showed that HOXA9 hypomethylation is significantly associated with the risk of CRC, and the molecular mechanism of HOXA9 hypomethylation-related carcinogenesis needs further study.

Significant association between KDM1A promoter hypomethylation and colorectal cancer in Han Chinese

Lysine-specific histone demethylase 1A gene (KDM1A) promotes tumorigenesis. The aim of this study was to investigate the association between KDM1A methylation and colorectal cancer (CRC). Currently, we collected 37 paired CRC tissues and adjacent non-tumor tissues from Jiangsu province and 75 paired CRC tissues and adjacent non-tumor tissues from Zhejiang province to conduct a two-stage experiment to study the association between KDM1A methylation and CRC. We used qMSP to measure the KDM1A promoter methylation, and the percentage of methylation reference (PMR) to quantify the KDM1A promoter methylation level. To investigate the effect of the selected KDM1A fragment on gene expression regulation, we also performed a dual luciferase reporter gene assay. In the stage I study, the KDM1A promoter methylation level in CRC tumor tissues was significantly lower than that in adjacent non-tumor tissues (median PMR: 6.93% vs 10.25%, P = 0.033). The results of the stage II study were similar to those of the stage I study (mean PMR: 12.94% versus 17.42%, P = 0.016). In addition, a clinical pathology subgroup analysis found that KDM1A hypomethylation was associated with CRC only in patients with well-differentiated CRC (stage I: P = 0.047; stage II: P = 0.040). The dual luciferase reporter assay showed that the transcriptional activity of the recombinant pGL3-KDM1A plasmid was significantly higher (fold change = 2, P = 0.0009). In conclusion, our results suggest that KDM1A hypomethylation is significantly associated with CRC.

Tumor p16INK4 gene expression and prognosis in colorectal cancer.

Hypermethylation of the tumor suppressor gene p16INK4 (p16) promoter is associated with worse prognosis in colorectal cancer (CRC). In the present study, it was investigated whether p16 mRNA expression correlates with the methylation of its promoter, and whether it influences prognosis in patients with CRC. DNA and RNA were extracted from 101 resected tumor specimens. A MethyLight assay was used to quantify p16 methylation in terms of percentage of methylated reference (PMR), and the expression of p16 mRNA was measured using reverse transcription‑polymerase chain reaction. Associations between p16 methylation or mRNA expression and patient survival were evaluated using Kaplan‑Meier analysis and Cox proportional hazards regression. p16 methylation was detected in 67 cases (66.3%) and the median PMR value was 0.344 (range, 0.00‑468.6). Using a cut‑off PMR value of 4, high p16 methylation was observed in 18 cases (17.8%). No significant association was observed between p16 methylation level and patient prognosis. As expected, a significant inverse association was observed between p16 methylation and mRNA expression (P=0.034). Amongst the 83 cases with low p16 methylation, a significantly worse outcome was identified in patients expressing high p16 mRNA expression levels (P=0.026). Multivariate analysis identified that p16 mRNA expression was an independent prognostic factor for worse survival (P=0.011). These results suggested a paradoxical association between high levels of p16 mRNA expression in the tumor and worse prognosis in patients with CRC.

Endothelial PAS domain protein 1 gene hypomethylation is associated with colorectal cancer in Han Chinese.

Endothelial PAS domain-containing protein 1 (EPAS1) serves a role in angiogenesis, which is important for the development of tumors, including colorectal cancer (CRC). The current study aimed to estimate whether EPAS1 methylation was associated with CRC. A two-stage association study of EPAS1 methylation and CRC was conducted. In the first phase, EPAS1 methylation was evaluated in the tumor and adjacent non-tumor tissue samples from 41 patients with sporadic CRC in Jiangsu province, China. The diagnostic value of methylation of EPAS1 for CRC in the second phase was evaluated in 79 patients with sporadic CRC and 22 normal individuals in Zhejiang province, China. The methylation assay was performed using a quantitative methylation-specific polymerase chain reaction (qMSP) method. The percentage of methylated reference (PMR) was used to quantify the methylation level. The first-stage results indicated that EPAS1 promoter methylation was significantly lower in CRC tumor tissues compared with 5-cm-para-tumor tissues (median PMR, 0.59 vs. 1.22%; P=0.027) and 10-cm-para-tumor tissues (median PMR, 0.59 vs. 1.89%; P=0.001). In addition, the second-stage results indicated that EPAS1 promoter methylation was significantly lower in tumor tissues compared with 5-cm-para-tumor tissues (median PMR, 1.91 vs. 6.25%; P=3×10−7) and normal intestinal tissues from healthy controls (median PMR, 1.91 vs. 28.4%; P=5×10−7). Receiver Operating Characteristic curve analysis of the second-stage data indicated that the highest area under the curve of EPAS1 hypomethylation was 0.851 between Zhejiang CRC tissues and Zhejiang normal intestinal tissues (sensitivity, 95.5%; specificity, 60.8%).

TNFRSF10C methylation is a new epigenetic biomarker for colorectal cancer.

BackgroundAbnormal methylation of TNFRSF10C was found to be associated with different types of cancers, excluding colorectal cancer (CRC). In this paper, the performance of TNFRSF10C methylation in CRC was studied in two stages.MethodThe discovery stage was involved with 38 pairs of CRC tumor and paired adjacent non-tumor tissues, and 69 pairs of CRC tumor and paired adjacent non-tumor tissues were used for the validation stage. Quantitative methylation specific PCR (qMSP) method and percentage of methylated reference (PMR) were used to test and represent the methylation level of TNFRSF10C, respectively. A dual-luciferase reporter gene experiment was conducted to evaluate the promoter activity of TNFRSF10C fragment.ResultsA significant association of TNFRSF10C promoter hypermethylation with CRC was found and validated (discovery stage: 24.67 ± 7.52 vs. 3.36 ± 0.89; P = 0.003; validation stage: 31.21 ± 12.48 vs. 4.52 ± 1.47; P = 0.0005). Subsequent analyses of TCGA data among 46 pairs of CRC samples further confirmed our findings (cg23965061: P = 4E − 6; cg14015044: P = 1E − 7). Dual-luciferase reporter gene assay revealed that TNFRSF10C fragment was able to significantly promote gene expression (Fold change = 2.375, P = 0.013). Our data confirmed that TNFRSF10C promoter hypermethylation can predict shorter overall survival of CRC patients (P = 0.032). Additionally, bioinformatics analyses indicated that TNFRSF10C hypermethylation was significantly associated with lower TNFRSF10C expression.ConclusionOur work suggested that TNFRSF10C hypermethylation was significantly associated with the risk of CRC.

MTHFD1 promoter hypermethylation increases the risk of hypertension

ABSTRACTObjectives: Methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) plays an essential role in folate-mediated one-carbon metabolism which determines both homocysteine remethylation and de novo thymidylate biosynthesis. Hyperhomocysteinemia is positively associated with essential hypertension. This study aimed to investigate the association of MTHFD1 promoter methylation with essential hypertension.Methods: Using the quantitative methylation-specific polymerase chain reaction (qMSP), the levels of MTHFD1 promoter methylation in 243 essential hypertension patients, 218 age- and gender-matched healthy controls. The relative changes in serum MTHFD1 promoter methylation were analyzed using the 2−ΔΔCt method. The percent of methylated reference (PMR) of MTHFD1 was used to evaluate the MTHFD1 promoter methylation levels.Results: In our current study, the MTHFD1 promoter methylation of hypertensive patients were both higher than the healthy control group (median PMR were 8.97% and 5.69%, respectively, all p < 0.001). Multivariable analysis showed MTHFD1 promoter hypermethylation increase the risk of essential hypertension (OR, 1.336; 95%CI, 1.235–1.446; p < 0.001). The area under the curve (AUC) of MTHFD1 promoter methylation was 0.739 in total patients with essential hypertension.Conclusions: MTHFD1 promoter hypermethylation was a potential biomarker for the diagnosis of essential hypertension.

Significant association of PRMT6 hypomethylation with colorectal cancer.

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. 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. 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. Our study firstly indicated that the hypomethylation of PRMT6 promoter could be a novel diagnostic biomarker for CRC.