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Oh, Tae Jeong,Oh, Hyun Il,Seo, Yang Yei,Jeong, Dongjun,Kim, Changjin,Kang, Hyoun Woo,Han, Yoon Dae,Chung, Hyun Cheol,Kim, Nam Kyu,An, Sungwhan BioMed Central 2017 CLINICAL EPIGENETICS Vol.9 No.1
<P><B>Background</B></P><P>Colorectal cancer (CRC) screening is the most efficient strategy to reduce disease-related mortality. Frequent aberrant DNA methylation is known to occur in selected genes and early during CRC development, which has emerged as a new epigenetic biomarker for early detection of CRC. Previously, we reported that we identified that CpG sites of <I>SDC2</I> were aberrantly methylated in tumor tissues of most CRC patients through comprehensive methylation analysis and demonstrated a high potential of quantification of <I>SDC2</I> methylation in blood for early detection of colorectal cancer. In this study, we aim to investigate the feasibility of quantifying <I>SDC2</I> methylation in stool DNA for the early detection of CRC. The objective of this study was to confirm a high frequency of <I>SDC2</I> methylation in tumor tissues at various stages of CRC and investigate the feasibility of a quantitative test for <I>SDC2</I> methylation in fecal DNA by highly sensitive and accurate real-time PCR for early detection of CRC.</P><P><B>Methods</B></P><P>Bisulfite-pyrosequencing assay was performed to measure the <I>SDC2</I> methylation status in tissue samples. For methylation analysis in stool DNA, a highly sensitive and accurate method was applied which implements consecutive two rounds of PCR consisting of unidirectional linear target enrichment (LTE) of <I>SDC2</I> and quantitative methylation-specific real time PCR (qMSP) for <I>SDC2</I>, named as me<I>SDC2</I> LTE-qMSP assay. Its limit of detection was 0.1% methylation (corresponding to ~ 6 copies in total ~ 6200 genome copies).</P><P><B>Results</B></P><P>Positive <I>SDC2</I> methylation was observed in 100% of primary tumors, 90.6% of adenomatous polyps, 94.1% of hyperplastic polyps, and 0% of normal tissues. <I>SDC2</I> methylation level also significantly (<I>P</I> < 0.01) increased according to the severity of lesions. In stool DNA test for <I>SDC2</I> methylation by LTE-qMSP comparing CRC patients with various stages (I to IV) (<I>n</I> = 50) and precancerous lesions (<I>n</I> = 21) with healthy subjects (<I>n</I> = 22), the overall sensitivity was 90.0% for detecting CRC and 33.3% for detecting small polyps, with a specificity of 90.9%.</P><P><B>Conclusions</B></P><P>Taken together, our result indicates that stool DNA-based <I>SDC2</I> methylation test by LTE-qMSP is a potential noninvasive diagnostic tool for early detection of CRC.</P>
Oh, Myungsok,Hoehn, Benjamin Douglass,Moon, Youngho,Oh, Taejeong,Ko, Youngbok,An, Sungwhan Elsevier 2012 The Journal of Molecular Diagnostics Vol.14 No.4
<P>Herein, we describe a novel multiplex genotyping method, GTPlex-PyroSeq. This method consists of two phases: multiplex PCR followed by a single reaction of pyrosequencing. This study demonstrates how GTPlex-PyroSeq can be adapted for the determination of multiple human papillomavirus (HPV) genotypes. A biotinylated consensus primer, GP6+, and 15 high-risk HPV type-specific primers are used for multiplex PCR. Each type-specific primer has a 5′-tag unique ID sequence connected to a pyrosequencing primer binding region. The unique ID sequence is composed of three parts: i) a single nucleotide ID representing a specific genotype; ii) a sign post; and iii) an end mark. This design allows multiple genotype determination under an ID sequence-dependent nucleotide dispensation order during pyrosequencing. Following initial studies using HPV plasmids and cell lines, we evaluated the clinical utility and effectiveness by comparing our assay with direct sequencing and HPV DNA chip analysis of 80 samples from high-risk, HPV-positive patients. We found in single-type infections, 100% concordance with direct sequencing (70 of 80 perfect matches) and 97.5% concordance with HPV DNA chip data (50 of 80 perfect matches). Additionally, our system was superior to direct sequencing in detection of multiple infections (12 of 80), with a limit of detection of 100 copies. The scalability of this multiplex system, with its open-platform design and ability to use various sample types, makes the GTPlex applicable for use in multiple settings.</P>
Unconventional immune cells in the gut mucosal barrier: regulation by symbiotic microbiota
Yoo Ji-Sun,Oh Sungwhan F. 생화학분자생물학회 2023 Experimental and molecular medicine Vol.55 No.-
The mammalian gut is the most densely colonized organ by microbial species, which are in constant contact with the host throughout life. Hosts have developed multifaceted cellular and molecular mechanisms to distinguish and respond to benign and pathogenic bacteria. In addition to relatively well-characterized innate and adaptive immune cells, a growing body of evidence shows additional important players in gut mucosal immunity. Among them, unconventional immune cells, including innate lymphoid cells (ILCs) and unconventional T cells, are essential for maintaining homeostasis. These cells rapidly respond to bacterial signals and bridge the innate immunity and adaptive immunity in the mucosal barrier. Here, we focus on the types and roles of these immune cells in physiological and pathological conditions as prominent mechanisms by which the host immune system communicates with the gut microbiota in health and diseases.
Genome-wide identification of OTP gene as a novel methylation marker of breast cancer.
Kim, Myung Soon,Lee, Jinsun,Oh, Taejeong,Moon, Youngho,Chang, Eilsung,Seo, Kwang Sun,Hoehn, Benjamin Douglas,An, Sungwhan,Lee, Jeung-Hoon National Hellenic Research Foundation 2012 ONCOLOGY REPORTS Vol.27 No.5
<P>Aberrant DNA methylation occurs early and frequently in tumorigenesis. Identification of DNA methylation biomarkers is a field that provides potential for improving the clinical process of breast cancer diagnosis. We utilized a genome-wide technique, methylated DNA isolation assay (MeDIA), in combination with high-resolution CpG microarray analysis to identify hypermethylated genes in breast cancer. Among differentially methylated genes between tumor and adjacent normal tissues, 3 candidate genes (LHX2, WT1 and OTP) were finally selected through a step-wise filtering process and examined for methylation status in normal tissues, primary tumor, and paired adjacent normal-appearing tissues from 39 breast cancer patients. Based on the calculated cut-off values, all genes showed significantly higher frequencies of aberrant hypermethylation in primary tumors (43.6% for LHX2, 89.7% for WT1 and 100% for OTP, p<0.05) while frequencies were intermediate in paired adjacent normal tissues and absent in normal tissues. On further analysis, the methylation level in primary tumors was not significantly correlated with clinicopathological features. Interestingly, DNA methylation of a novel gene OTP was detected in adjacent normal tissues even 6?cm away from primary tumors, suggesting that OTP methylation may qualify as a biomarker for the early detection of breast cancer. In conclusion, we successfully identified a novel gene OTP frequently methylated in breast cancer by genome-wide screening. Our results suggest that the OTP gene may play a crucial role in breast carcinogenesis, although further clinical validation will be needed to evaluate the potential application of OTP in the early detection of breast cancer.</P>
The Role of Vimentin as a Methylation Bio-marker for Early Diagnosis of Cervical Cancer
Samil Jung,Lisha Yi,김진선,정동준,Taejeong Oh,Chang-Hwan Kim,Chang-Jin Kim,Jin Shin,Sungwhan An,이명석 한국분자세포생물학회 2011 Molecules and cells Vol.31 No.5
Multiple cytosine guanine dinucleotides (CpG island) are found in the VIM promoter region. The levels of VIM pro-moter methylation and VIM gene expression were investi-gated in 7 cervical cancer cell lines and 50 human tissue samples with a distinctive degree of malignant trans-for-mation. While multiple CpG sites in the VIM promoter were highly methylated in CIN III and invasive carcinoma cells, they were rarely methylated in normal cells. Our result shows that methylation in the VIM promoter appears to start from CIN I and CIN II, relatively early stages of multi-step carcinogenesis. This epigenetic alteration in VIM promoter suggests the availability as a biomarker for the early diagnosis and prevention of cervical cancer. We also show that hypermethylation in the VIM promoter is re-sponsible for transcriptional silencing of the VIM gene in cervical cancer cells. In addition, our result shows that exogenous overexpression of the VIM gene in SiHa cer-vical cancer cells slightly activated cell proliferation and migration as shown in soft agar colony formation and migration assays.
Yeo, Min‐,Kyung,Liang, Zhe Long,Oh, Taejeong,Moon, Youngho,An, Sungwhan,Kim, Min Kyeong,Kim, Koon Soon,Shong, Minho,Kim, Jin‐,Man,Jo, Young Suk Blackwell Publishing Ltd 2011 Clinical endocrinology Vol.75 No.4
<P><B>Summary</B></P><P><B>Context </B> Recently, tremendous efforts have been made towards the development of sensitive techniques to detect the BRAF<SUP>V600E</SUP> mutation in fine needle aspiration biopsy (FNAB) samples. However, newly developed quantitative and semi‐quantitative methods, such as dual‐priming oligonucleotide (DPO)‐based multiplex polymerase chain reaction (PCR), have the potential to generate false‐positive (FP) results.</P><P><B>Objectives </B> To eliminate the possibility of FP results, we generated a receiver operating characteristic (ROC) curve to investigate the diagnostic accuracy of pyrosequencing using quantitative data.</P><P><B>Design </B> Cytological diagnoses of 983 thyroid nodules were made according to the Bethesda System 2007. The BRAF<SUP>V600E</SUP> mutation was analysed by pyrosequencing, and statistical analyses were performed.</P><P><B>Results </B> Of the 983 nodules, 902 were adopted to evaluate the diagnostic value of pyrosequencing. The number of pathologically confirmed malignancies was 192, of which 182 were papillary thyroid cancer (PTC). By generating an ROC curve, we defined the optimal cut‐off value of the mutant allele peak as 5·95% (area under the curve, 0·849; sensitivity, 0·55; 1‐specificity, 0). When we applied this selective cut‐off value, the number of PTCs positive for BRAF<SUP>V600E</SUP> was 99 (54·4% of the total number of PTCs). With cytology alone, the diagnostic sensitivity and specificity of detecting malignancy were 71·2% and 100%, respectively. Pyrosequencing improved the diagnostic sensitivity from 71·2% to 78·5% (McNemar’s test, <I>P</I> < 0·001), without any change in the diagnostic specificity. When ‘suspicious for malignancy’ was considered a positive cytological outcome, pyrosequencing increased the diagnostic sensitivity of cytology from 95·8% to 96·9%; however, this improvement did not show statistical significance (McNemar’s test, <I>P</I> > 0·05).</P><P><B>Conclusions </B> Pyrosequencing is an effective method for detecting the BRAF<SUP>V600E</SUP> mutation in FNAB samples. By allowing the optimal cut‐off value to be determined, pyrosequencing improves the diagnostic sensitivity while eliminating the possibility of FP results.</P>