A Novel Method for Multiplex Genotyping in a Single Reactor Using GTPlex-PyroSeq : Genotyping HPV as a Prototype

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>

Genome-wide analysis of epigenetic changes in mouse sperm by maternal exposure to endocrine disruptor vinclozolin

이승훈,TaeJeong Oh 대한독성 유전단백체 학회 2012 Molecular & cellular toxicology Vol.8 No.1

The genome-wide epigenetic alterations (DNA methylation) in germ cells have not been extensively studied in mice by exposure to environmental hazards. We first report here that a genome-wide analysis of CpG methylation in F1 mice sperm by maternal exposure to the anti-androgenic compound vinclozolin (VCZ). The gestated (GD) female mice (n=8) were injected intraperitoneally with VCZ (100 mg/kg/day), from GD 8 to GD 15 to induce reproductive toxicity and epigenetic alterations in mice. Our observations showed a loss of normal spermatogenesis and abnormal seminiferous tubule morphology in VCZ-treated F1 males. We also confirmed reduced fertility of the VCZ-exposed F1 male mice and increased abnormality in F2 fetuses. We compared the methylation patterns of five untreated with 13 VCZ-treated F1 mice male via high-resolution CpG microarray analysis to determine CpG methylation alterations in F1 male sperm DNA. The overall methylation between control and treated F1 sperm showed normal distribution, and differentially methylated CpG islands were frequently mapped between the -0.5 kb and +1.0 kb regions of the transcription start site (TSS). We identified more than several hundred epigenetically altered genes by statistical analysis (P⁄0.05). More than 50% of the statistically significantly methylated loci were also mapped at the vicinity of TSS. We further examined the effects of VCZ on the mouse repeated elements and paternally imprinted genes. VCZ induced no epigenetic alterations in repeated mouse elements (LINE and B1) and the paternally imprinted Dlk1 gene, whereas it did affect the paternally imprinted H19 gene. In conclusion, we successfully explored VCZ-induced genome-wide epigenetic changes on F1 mice sperm DNA by global methylation profiling, and our data will provide important clues for studying the epigenetic changes of germ cells due to exposure to environmental hazards.

Comparative Analysis of Growth-Phase-Dependent Gene Ex-pression in Virulent and Avirulent Streptococcus pneumoniae Using a High-Density DNA Microarray

고관수,Sulhee Park,오원섭,Ji-Yoeun Suh,TaeJeong Oh,Sungwhan Ahn,천종식,송재훈 한국분자세포생물학회 2006 Molecules and cells Vol.21 No.1

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.

Pyrosequencing cut‐off value identifying BRAF^V600E mutation in fine needle aspiration samples of thyroid nodules

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>

독거노인의 사회서비스 욕구 유형 및 특성에 관한 연구

이정은(Jungeun Lee),류진아(Jina Ryoo),하태정(Taejeong Ha),오다은(Daeun Oh) 충남대학교 사회과학연구소 2021 사회과학연구 Vol.32 No.3

The purpose of this study is to identify the types of social service needs of older adults living alone, and to examine the characteristics and various predictive factors of each type. In order to achieve these study objectives, 552 single households aged 65 and over were analyzed from the data of the 2019 Social Service Demand and Supply Survey. First, we conducted latent class analysis to classify latent groups representing similar patterns of social service needs. Multinomial logistic regression was then conducted to identify the characteristics and influencing factors of each group type. As a result, the latent groups of older adults living alone considering the type of social service needs were classified into three groups: health/social participation/leisure-oriented cluster (8.7%), health/adult care/residential environment-oriented cluster (22.1%), and health-oriented cluster (69.1%). Results of multinomial logistic analysis revealed that the common factor separating the ‘health-oriented cluster’ and the other two groups was the subjective economic level. Based on this study, various policy interventions need to be implemented based on the complex needs of older adults living alone. In addition, this study can be used as a basis for designing customized services for integrated case management of older adults living alone.

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>

과학기술혁신 정책진단 프레임워크 구축

이정우(Jungwoo Lee),박기범(Kibeom Park),황석원(Seogwon Hwang),하태정(Taejeong Ha),안두현(Doohyun Ahn),양현채(Hyeonchae Yang),이혁(Hyeok Lee),목은지(Eunji Mok),하리다(Reeda Ha),김지선(Jiseon Kim),오승환(Seunghwan Oh),여영준(Yeongjun Yeo 과학기술정책연구원 2021 정책연구 Vol.- No.-

The role of vimentin as a methylation biomarker for early diagnosis of cervical cancer

Jung, Samil,Yi, Lisha,Kim, Jinsun,Jeong, Dongjun,Oh, Taejeong,Kim, Chang-Hwan,Kim, Chang-Jin,Shin, Jin,An, Sungwhan,Lee, Myeong-Sok Springer-Verlag 2011 Molecules and cells Vol.31 No.5

Combination Analysis of PCDHGA12 and CDO1 DNA Methylation in Bronchial Washing Fluid for Lung Cancer Diagnosis

Park Se Jin,Kang Daeun,Lee Minhyeok,Lee Su Yel,Park Young Gyu,Oh TaeJeong,Jang Seunghyun,Hwang Wan Jin,Kwon Sun Jung,An Sungwhan,Son Ji Woong,Jeong In Beom 대한의학회 2024 Journal of Korean medical science Vol.39 No.2

Background: When suspicious lesions are observed on computer-tomography (CT), invasive tests are needed to confirm lung cancer. Compared with other procedures, bronchoscopy has fewer complications. However, the sensitivity of peripheral lesion through bronchoscopy including washing cytology is low. A new test with higher sensitivity through bronchoscopy is needed. In our previous study, DNA methylation of PCDHGA12 in bronchial washing cytology has a diagnostic value for lung cancer. In this study, combination of PCDHGA12 and CDO1 methylation obtained through bronchial washing cytology was evaluated as a diagnostic tool for lung cancer. Methods: A total of 187 patients who had suspicious lesions in CT were enrolled. PCDHGA12 methylation test, CDO1 methylation test, and cytological examination were performed using 3-plex LTE-qMSP test. Results: Sixty-two patients were diagnosed with benign diseases and 125 patients were diagnosed with lung cancer. The sensitivity of PCDHGA12 was 74.4% and the specificity of PCDHGA12 was 91.9% respectively. CDO1 methylation test had a sensitivity of 57.6% and a specificity of 96.8%. The combination of both PCDHGA12 methylation test and CDO1 methylation test showed a sensitivity of 77.6% and a specificity of 90.3%. The sensitivity of lung cancer diagnosis was increased by combining both PCDHGA12 and CDO1 methylation tests. Conclusion: Checking DNA methylation of both PCDHGA12 and CDO1 genes using bronchial washing fluid can reduce the invasive procedure to diagnose lung cancer.