The Identification and Validation of EphA7 Hypermethylation, a Novel Biomarker, in Cervical Cancer

Background Aberrant methylation of EphA7 has been reported in the process of carcinogenesis,but not including cervical cancer.Therefore,an integration study was performed to explore the association between EphA7 hypermethylation and cervical cancer and validate the potential value of EphA7 hypermethylation in the diagnosis of cervical cancer. Results Here, we performed an integration study to identify and validate the association between EphA7 methylation and cervical cancer. First, data on EphA7 methylation and expression in cervical cancer were extracted and analyzed via bioinformatics tools. The results showed that EphA7 promoter methylation levels were significantly increased in cervical cancer compared to normal tissues (P<0.001) and negatively correlated with EphA7 expression. Subsequently, these prediction results were confirmed in cell lines; moreover, CRISPR-based methylation perturbation tools (dCas9-Tet1/DNMT3a) were constructed to further demonstrate that DNA methylation participates in the regulation of EphA7 expression directly. Ultimately, the clinical value of EphA7 methylation in cervical cancer was validated in cervical tissues and Thinprep cytologic test (TCT) samples by methylation-specific PCR (MSP) and quantitative methylation-specific PCR (QMSP), respectively. Consistently, the methylation level and the positive rate of EphA7 gradually increased with severity from normal to cancer stages in TCT samples (P<0.01). Conclusion Above all, EphA7 presents hypermethylation in the cervical cancer,which is a potential biomarker in the diagnosis of cervical cancer.

Development and Performance Evaluation of TaqMan Real-time Fluorescence Quantitative Methylation Specific PCR for Detecting Methylation Level of PER2

Background: PER2 gene methylation is closely related to the occurrence and progress of some cancers, but there is no method to quantitatively detect PER2 methylation in conventional laboratories. So, we established a TaqMan real-time fluorescence quantitative methylation specific PCR (TaqMan real-time FQ-MSP) assay and use it for quantitative detection of PER2 methylation in leukemia patients. Methods: According to the PER2 sequence searched by GenBank, a CpG sequence enrichment region of the PER2 gene promoter was selected, and the methylated and unmethylated target sequences were designed according to the law of bisulfite conversion of DNA to construct PER2 methylation positive and negative reference materials. Specific primers and probe were designed. The reference materials were continuously diluted into gradient samples by 10-fold ratio to evaluate the analytical sensitivity, specificity,accuracy and reproducibility of the method, and the analytical sensitivity of TaqMan real-time FQ-MSP assay was compared with that of the conventional MSP assay. At the same time, the new-established TaqMan real-time FQ-MSP assay and the conventional MSP assay were used to detect the PER2 methylation level of 81 patients with leukemia, and the samples with inconsistent detection results of the two assays were sent to pyromethylation sequencing to evaluate the clinical detection performance .Results: The minimum detection limit of TaqMan real-time FQ-MSP assay for detecting PER2 methylation level established in this study was 6 copies/uL, and the coefficient of variation(CV) of intra-assay and inter-assay was less than 3%. Compared with the conventional MSP assay, it has higher analytical sensitivity. For the samples with inconsistent detection results, the results of pyrosequencing and TaqMan real-time FQ-MSP assay are consistent. Conclusion: TaqMan real-time FQ-MSP assay of PER2 methylation established in this study has high detection performance and can be used for the detection of clinical samples.

Case Report: An Atypical Angelman Syndrome Case With Obesity and Fulfilled Autism Spectrum Disorder Identified by Microarray

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders which are etiologically heterogeneous. Chromosomal microarray is now recommended as the first-tier clinical diagnostic test for ASD. We performed chromosomal microarray in 16 Thai patients with ASD using an Illumina HumanCytoSNP-12 v2.1 array and found one case with uniparental disomy (UPD) of chromosome 15. Methylation-specific PCR showed abnormal methylation of the maternal SNRPN allele. Haplotype analysis revealed that the patient had received both chromosomes 15 from his father. These results were consistent with Angelman syndrome. However, his clinical features had no clinical significance for classic Angelman syndrome. He had first presented at the pediatric clinic with no speech, poor social interaction skills and repetitive behaviors consistent with ASD based on the DSM-IV criteria at 2 years of age and later confirmed by ADOS at 5 years of age. He was strikingly overweight but had no dysmorphic facies, seizures nor ataxia and was diagnosed as non-syndromic ASD, a diagnosis which was believed until at 10 years of age, his DNA was included for analysis in this current cohort study. Our findings suggest that ASD patients with unknown etiology should be considered for methylation-specific PCR testing for Angelman syndrome where chromosomal microarray is not available. In the study, we also review the clinical features of Angelman syndrome caused by UPD and the frequency of ASD in individuals with Angelman syndrome.

Assessment of Aberrant Promoter Hypermethylation of RASSF1a and P16 in Endometrial Carcinoma

Endometrial carcinoma is one of the most common gynecological malignancies known to affect around 142000 women worldwide. Aberrant promoter hypermethylation of tumor suppressor genes is a common epigenetic alteration reported in cancers. In the present study we report the aberrant promoter hypermethylation of RASSF1a and p16 in 78 endometrial cancer patients. Methods: Endometrial carcinoma samples were collected after pathological examination and DNA was extracted. The DNA was subjected to sodium bisulfite modification and then used to perform methylation specific PCR. The PCR was performed using a two step procedure of nested and methylation specific PCR. The PCR product was visualized using agarose gel electrophoresis. The results obtained were correlated with various clinicopathological parameters. Chi square test was used for statistical analysis and a p-value of <0.05 was considered statistically significant. Result: 60 of the 78 (76.92%) samples showed methylation for RASSF1a gene. 28 of the 78 (35.8%) samples showed methylation for p16 gene. A higher methylation frequency was observed for RASSF1a in the endometrioid and poorly differentiated histological subtypes and stage I and II of the disease. Conclusion: Aberrant promoter hypermethylation of RASSF1a and p16 is known to play an important role in endometrial cancer initiation and progression. The methylation patterns can be used as an important molecular marker for early diagnosis and prognosis of endometrial cancer. Keywords: Epigenetic alterations, DNA Hypermethylation, Endometrial cancer, RASSF1a, p16, Methylation-specific PCR.

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.

SOMATOSTATIN (SST) PROMOTER HYPERMETHYLATION IN ASSOCIATION WITH COLORECTAL CANCER

Colorectal cancer (CRC) is one of the most common diagnosis malignancies with different risk factors, including environmental and genetic. Several genes, called tumor suppressor genes, play an essential role in inhibiting these risk factors by preventing tumor development. One of these genes is somatostatin (SST). Somatostatin is an antiproliferative peptide with pro-apoptotic effects that enhance cell death to prevent tumor growth. This study aimed to investigate the association relationship between DNA methylation in SST promotor and colorectal cancer progression. After DNA bisulfite conversion, SST promoter methylation was examined using quantitative methylation‐specific PCR (qMSP) in 71 cases (19 metastasis CRC, 28 early-stage CRC, and 24 healthy controls). Quantitative methylation‐specific PCR (qMSP) is a real-time PCR method used to determine the unmethylated and methylated cytosine residues using a specific set of primers. The percentage of hypermethylation in SST promoter was 17%, 60%, and 79% for healthy controls, early-stage, and metastasis CRC groups. The results showed a significant association between DNA hypermethylation of SST promoter and CRC progression. P-values were 0.0364 for the early-stage group and 0.0138 for the metastasis group. The results also supported that the DNA hypermethylation block the expression of SST, which in turn induce carcinogenesis. The detection of SST promoter hypermethylation at early stage of cancer could be used as a biomarker for screening and prognosis of CRC.