LEPR hypomethylation is significantly associated with gastric cancer in males.

BACKGROUND: Previous 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 METHODS: Tumor 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). RESULTS: Our 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%. CONCLUSION: LEPR 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.

GPX3 hypermethylation in gastric cancer and its prognostic value in patients aged over 60.

AIM: This study investigated the association between GPX3 methylation and gastric cancer (GC), and explored its prognostic value in patients undergoing radical gastrectomy. MATERIALS & METHODS: The methylation levels of tumor and paracancerous tissues were detected by quantitative methylation-specific PCR method. RESULTS: GPX3 was hypermethylated in GC (p = 4E-4), and was specific for patients with lymphatic metastasis (+), tumor invasion depth >3 cm and patients with poor differentiation. Additionally, GPX3 hypermethylation predicts a tumor recurrence in patients aged >60 (p = 0.019). Data from The Cancer Genome Atlas (TCGA) further confirmed GPX3 hypermethylation (cg21504918: -0.08 vs -0.25, p = 0.001). Additionally, TCGA showed an inverse correlation between GPX3 methylation and expression (p = 7E-18, r = -0.427). Data analysis of Gene Expression Omnibus (GEO) database showed that 5-aza-2'-deoxycytidine demethylating agent increased GPX3 expression (fold-change >2.19, p = 0.001). CONCLUSION: Our results indicated GPX3 hypermethylation in GC, and predicted a shorter tumor recurrence time in patients aged >60.

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.

The impact of heat waves on the mortality of Chinese population: A systematic review and meta-analysis.

BACKGROUND: Many studies had shown that with global warming, heat waves may increase the mortality risk of Chinese populations. However, these findings are not consistent. Therefore, we elucidated the associations by meta-analysis and quantified the magnitude of these risks, as well as the underlying factors. METHODS: We searched the China National Knowledge Infrastructure (CNKI), Wanfang database, PubMed, EMBASE, and Web of Science for literature screening up to Nov 10, 2022, to analyze the effect of heat waves on mortality in the Chinese population. Literature screening and data extraction were performed independently by two researchers and the data were merged by meta-analysis. In addition, we conducted subgroup analysis by sex, age, years of education, region, and number of events to explore the source of heterogeneity. RESULTS: Fifteen related studies on the impact on heat waves of the death of Chinese people were included in this study. The results of the meta-analysis showed that heat waves were significantly associated with increased mortality from non-accidental deaths, cardiovascular diseases, stroke, respiratory diseases, and circulatory diseases in the Chinese population: non-accidental mortality (RR = 1.19, 95% CI: 1.13-1.27, P < .01), cardiovascular diseases (RR = 1.25, 95% CI: 1.14-1.38), stroke (RR = 1.11, 95% CI: 1.03-1.20), respiratory diseases (RR = 1.18, 95% CI: 1.09-1.28), and circulatory diseases (RR = 1.11, 95% CI: 1.06-1.17). Subgroup analyses showed that heat waves had a higher risk of non-accidental death for those with <6 years of education than for those with ≥6 years of education. Meta-regression analysis showed that the contribution of the study year to the inter studied heterogeneity was 50.57%. The sensitivity analysis showed that the exclusion of any single study did not materially alter the overall combined effect. The meta-analysis method indicated no obvious evidence of publication bias. CONCLUSIONS: The results of the review indicated that heat waves were associated with increased mortality in the Chinese population, that attention should be paid to high-risk groups, and that public health policies and strategies should be implemented to more effectively respond to and adapt to climate change.

Genome-wide identification and characterization of 14-3-3 gene family related to negative regulation of starch accumulation in storage root of Manihot esculenta.

The 14-3-3 protein family is a highly conservative member of the acid protein family and plays an important role in regulating a series of important biological activities and various signal transduction pathways. The role of 14-3-3 proteins in regulating starch accumulation still remains largely unknown. To investigate the properties of 14-3-3 proteins, the structures and functions involved in starch accumulation in storage roots were analyzed, and consequently, 16 Me14-3-3 genes were identified. Phylogenetic analysis revealed that Me14-3-3 family proteins are split into two groups (ε and non-ε). All Me14-3-3 proteins contain nine antiparallel α-helices. Me14-3-3s-GFP fusion protein was targeted exclusively to the nuclei and cytoplasm. In the early stage of starch accumulation in the storage root, Me14-3-3 genes were highly expressed in high-starch cultivars, while in the late stage of starch accumulation, Me14-3-3 genes were highly expressed in low-starch cultivars. Me14-3-3 I, II, V, and XVI had relatively high expression levels in the storage roots. The transgenic evidence from Me14-3-3II overexpression in Arabidopsis thaliana and the virus-induced gene silencing (VIGS) in cassava leaves and storage roots suggest that Me14-3-3II is involved in the negative regulation of starch accumulation. This study provides a new insight to understand the molecular mechanisms of starch accumulation linked with Me14-3-3 genes during cassava storage root development.

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.

FANCF hypomethylation is associated with colorectal cancer in Han Chinese.

BACKGROUND/AIMS: 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). MATERIALS AND METHODS: 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. RESULTS: 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). CONCLUSION: 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.

PTPN11 hypomethylation is associated with gastric cancer progression.

Protein tyrosine phosphatase non-receptor type 11 (PTPN11) encodes the tyrosine phosphatase SHP-2 that is overexpressed in gastric cancer (GC). In the present study, the association of PTPN11 methylation levels with the incidence of GC and its correlation with SHP-2 overexpression were investigated. The methylation levels of PTPN11 in tumor and adjacent normal tissues of 112 GC patients were assessed by quantitative methylation specific PCR (qMSP). The Cancer Genome Atlas (TCGA) public database was used to analyze the association between PTPN11 methylation and PTPN11 expression. Survival analyses were conducted in order to evaluate the prognostic value of PTPN11 methylation for GC. The results of the qMSP analysis indicated that the methylation levels of PTPN11 in GC tumor tissues were significantly decreased compared with those noted in the normal adjacent tissues (mean with standard deviation: 40.91±26.33 vs. 51.99±37.37, P=0.007). An inverse correlation between PTPN11 methylation levels and PTPN11 mRNA expression levels (P=4×10-6, r=-0.237) was noted. Subgroup analyses indicated that the association of PTPN11 hypomethylation with the incidence of GC was specific to male subjects (P=0.015), heavy drinking patients (P=0.019), patients with poor tumor differentiation (P=0.010) and patients with tumor node and metastasis (TNM) stage III+IV (P=0.008). Kaplan-Meier analyses and log-rank test suggested that PTPN11 hypomethylation was not associated with GC patient overall survival (P=0.605) and recurrence (P=0.485), although it could predict the recurrence of GC patients up to and including 60 years (≤60, P=0.049). The results indicated that PTPN11 levels were hypomethylated in GC patients. TCGA data analysis suggested that PTPN11 hypomethylation could cause an upregulation in the transcription levels of PTPN11. Although, this may explain the pattern of SHP-2 overexpression in GC, additional studies are required to verify this hypothesis. The association of PTPN11 hypomethylation with GC incidence may be specific to male patients, heavy drinking patients, patients with poor tumor differentiation and patients with TNM stage of III+IV. PTPN11 hypomethylation can be considered a biomarker for the recurrence of GC patients with an age of 60 years or lower.

APOE hypermethylation is significantly associated with coronary heart disease in males.

APOE is involved in exogenous and endogenous lipid metabolism and is a candidate gene for coronary heart disease (CHD). Using the SYBR-Green quantitative methylation-specific PCR method, we measured APOE methylation levels in 640 cases of CHD and 436 controls. Meanwhile, we used the Sequenom MassARRAY platform to genotype APOE rs7412 and rs7259620. In men, we found that CHD patients had significantly lower levels of APOE methylation than non-CHD controls (P = 0.044). In addition, rs7412-T and rs7259620-A were protective factors for CHD in males (rs7412-T: OR = 0.527, allele P = 0.004; rs7259620-A: OR = 0.743, allele P = 0.029). In the CHD group and the non-CHD control group, APOE methylation levels were inversely correlated with age (total patients: r = -0.015, P = 0.009, total controls: r = -0.185, P = 1E-04). In the female control group, the APOE methylation levels were inversely correlated with plasma APOA1 protein levels (female controls: r = -0.188, P = 0.008). In the total controls and female controls, APOE methylation levels were inversely correlated with plasma APOE protein levels (total controls: r = -0.110, P = 0.031, female controls: r = -0.188, P = 0.008). Our GDMR interaction analysis showed that there was an interaction between APOE methylation and rs7412 (P = 0.011). In addition, there was a significant interaction between APOE methylation, rs7259620, gender, smoking, LDL, TC, and APOE protein levels in the risk of CHD (P = 0.011). In summary, our research showed that the methylation of APOE was important for the risk of CHD in men.

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.

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.

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.

TNFRSF10C methylation is a new epigenetic biomarker for colorectal cancer.

BACKGROUND: Abnormal 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. METHOD: The 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. RESULTS: A 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. CONCLUSION: Our work suggested that TNFRSF10C hypermethylation was significantly associated with the risk of 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%).

Diagnostic value of WIF1 methylation for colorectal cancer: a meta-analysis.

As a common antagonist of Wnt/ß-catenin signaling, Wnt inhibitory factor 1 (WIF1) plays an important role in the tumor progression. The aim of our meta-analysis was to summarize the diagnostic value of WIF1 methylation in colorectal cancer (CRC). Eligible studies were retrieved by a systemic search among PubMed, Embase, CNKI, and Wanfang literature databases. The diagnostic value of WIF1 methylation for CRC was assessed by the summary receiver operating characteristics (SROC) test. Our meta-analysis of 12 studies between 1420 CRC samples and 946 control samples showed that WIF1 hypermethylation was significantly associated with CRC (P < 0.001, OR = 30.10, 95% CI = 19.48-46.50). WIF1 hypermethylation, as a diagnostic biomarker for CRC, has a pooled sensitivity of 0.40 (95% CI: 0.37-0.42), a pooled specificity of 0.95 (95% CI: 0.93-0.96), a pooled positive-likelihood ratio (PLR) of 8.65 (95% CI, 4.47-16.73), and a pooled negative-likelihood ratio (NLR) of 0.41 (95% CI, 0.30-0.55), a diagnostic odds ratio (DOR) of 26.86 (95% CI: 15.73-45.89), and an area under the curve (AUC) of 0.9115. In conclusion, our study established that WIF1 hypermethylation might be a promising diagnostic biomarker for CRC.

APOE hypermethylation is associated with autism spectrum disorder in a Chinese population.

Abnormal apolipoprotein E (APOE) methylation has been demonstrated to be associated with Alzheimer's disease, which may have overlapping mechanisms with autism spectrum disorder (ASD). Thus, the purpose of the present study was to assess the possible link between APOE methylation and ASD. Genomic DNA was extracted from peripheral blood and subjected to a methylation assay. SYBR green-based quantitative methylation-specific polymerase chain reaction analysis was used to measure APOE methylation in 62 pediatric patients with ASD and 73 age-matched healthy subjects. The APOE methylation in each sample was expressed as a percentage of methylation of a reference (PMR). The results indicated that APOE methylation in pediatric patients with ASD was significantly higher than that in the healthy controls (median PMR, 33 vs. 11%; P=2.36×10-10). Receiver operating characteristic curve demonstrated that PMR of 15.4% was the optimal cut-off for predicting ASD (area under curve, 0.817; sensitivity, 93.5%; specificity, 72.6%; P=2.36×10-10). In summary, the present results indicated that APOE hypermethylation in peripheral blood DNA may be used as a diagnostic biomarker for ASD.

Association between the methylation of six apoptosis­associated genes with autism spectrum disorder.

Excessive apoptosis hinders the process of brain maturation and is regarded as one of the principal risk factors for the development of autism spectrum disorder (ASD). The aim of the present study was to investigate the association between the methylation of six apoptosis­associated genes [transforming growth factor ß 1 (TGFB1), BCL2 associated X, apoptosis regulator, insulin like growth factor binding protein 3, protein kinase C ß 1, presenilin 2 and C­C motif chemokine ligand 2] and ASD. Using quantitative methylation­specific polymerase chain reaction technology, DNA methylation levels were detected in 42 autistic and 26 control subjects. The logistic regression analysis results demonstrated that of the six genes, only TGFB1 was significantly hypomethylated in peripheral blood samples from children with autism compared with control samples (mean percentage of methylated reference, 0.011% vs. 0.019%; age­adjusted P=0.028). In addition, TGFB1 methylation was identified to be positively associated with the interaction ability score from the Autism Behavior Checklist (r=0.452; P=0.035). These data suggested that decreased TGFB1 methylation may contribute to the development of ASD.