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Evangelina R, Ganesan S, George M. The Epigenetic Landscape: From Molecular Mechanisms to Biological Aging. Rejuvenation Res 2025; 28:93-112. [PMID: 40094262 DOI: 10.1089/rej.2024.0102] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/19/2025] Open
Abstract
Epigenetics, the study of heritable changes in gene expression that do not involve alterations to the deoxyribonucleic acid (DNA) sequence, plays a pivotal role in cellular function, development, and aging. This review explores key epigenetic mechanisms, including DNA methylation (DNAm), histone modifications, chromatin remodeling, RNA-based regulation, and long-distance chromosomal interactions. These modifications contribute to cellular differentiation and function, mediating the dynamic interplay between the genome and environmental factors. Epigenetic clocks, biomarkers based on DNAm patterns, have emerged as powerful tools to measure biological age and predict health span. This article highlights the evolution of epigenetic clocks, from first-generation models such as Horvath's multi-tissue clock to advanced second- and third-generation clocks such as DNAGrimAge and DunedinPACE, which incorporate biological parameters and clinical biomarkers for precise age estimation. Moreover, the role of epigenetics in aging and age-related diseases is discussed, emphasizing its impact on genomic stability, transcriptional regulation, and cellular senescence. Epigenetic dysregulation is implicated in cancer, genetic disorders, and neurodegenerative diseases, making it a promising target for therapeutic interventions. The reversibility of epigenetic modifications offers hope for mitigating age acceleration and enhancing health span through lifestyle changes and pharmacological approaches.
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Affiliation(s)
- Rachel Evangelina
- Centre for Clinical Pharmacology, SRM Medical College, Hospital and Research Centre, Kattankulathur, Tamil Nadu, India
| | - Subhashree Ganesan
- Centre for Clinical Pharmacology, SRM Medical College, Hospital and Research Centre, Kattankulathur, Tamil Nadu, India
| | - Melvin George
- Centre for Clinical Pharmacology, SRM Medical College, Hospital and Research Centre, Kattankulathur, Tamil Nadu, India
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Esman A, Salamaikina S, Kirichenko A, Vinokurov M, Fomina D, Sikamov K, Syrkina A, Pokrovskaya A, Akimkin V. Promoter Methylation of HIV Coreceptor-Related Genes CCR5 and CXCR4: Original Research. Viruses 2025; 17:465. [PMID: 40284908 PMCID: PMC12030890 DOI: 10.3390/v17040465] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2025] [Revised: 03/03/2025] [Accepted: 03/06/2025] [Indexed: 04/29/2025] Open
Abstract
The persistence of human immunodeficiency virus (HIV) within viral reservoirs poses significant challenges to eradication efforts. Epigenetic alterations, including DNA methylation, are potential factors influencing the latency and persistence of HIV. This study details the development and application of techniques to assess CpG methylation in the promoter regions of the CCR5 and CXCR4 genes, which are key HIV-1 coreceptors. Using both Sanger sequencing and pyrosequencing methods, we examined 51 biological samples from 17 people living with HIV at three time points: baseline (week 0) and post-antiretroviral therapy (ART) at weeks 24 and 48. Our results revealed that CXCR4 promoter CpG sites were largely unmethylated, while CCR5 promoter CpGs exhibited significant variability in methylation levels. Specifically, CCR5 CpG 1 showed a significant decrease in methylation from week 0 to week 48, while CXCR4 CpG 3 displayed a significant decrease between week 0 and week 24. These differences were statistically significant when compared with non-HIV-infected controls. These findings demonstrate distinct methylation patterns between CCR5 and CXCR4 promoters in people living with HIV over time, suggesting that epigenetic modifications may play a role in regulating the persistence of HIV-1. Our techniques provide a reliable framework for assessing gene promoter methylation and could be applied in further research on the epigenetics of HIV.
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Affiliation(s)
- Anna Esman
- Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Central Research Institute of Epidemiology, 111123 Moscow, Russia
| | - Svetlana Salamaikina
- Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Central Research Institute of Epidemiology, 111123 Moscow, Russia
| | - Alina Kirichenko
- Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Central Research Institute of Epidemiology, 111123 Moscow, Russia
| | - Michael Vinokurov
- Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Central Research Institute of Epidemiology, 111123 Moscow, Russia
| | - Darya Fomina
- Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Central Research Institute of Epidemiology, 111123 Moscow, Russia
- State Research Center—Burnazyan Federal Medical Biophysical Center of Federal Medical Biological Agency, 123098 Moscow, Russia
| | - Kirill Sikamov
- Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Central Research Institute of Epidemiology, 111123 Moscow, Russia
| | - Arina Syrkina
- Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Central Research Institute of Epidemiology, 111123 Moscow, Russia
| | - Anastasia Pokrovskaya
- Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Central Research Institute of Epidemiology, 111123 Moscow, Russia
- Medical Institute, Peoples’ Friendship University of Russia (RUDN University), 117198 Moscow, Russia
| | - Vasily Akimkin
- Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Central Research Institute of Epidemiology, 111123 Moscow, Russia
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Belinda A, Humardani FM, Dwi Putra SE, Widyadhana B. The potential of circulating free DNA of methylated IGFBP as a biomarker for type 2 diabetes Mellitus: A Comprehensive review. Clin Chim Acta 2025; 567:120104. [PMID: 39706247 DOI: 10.1016/j.cca.2024.120104] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2024] [Revised: 12/17/2024] [Accepted: 12/17/2024] [Indexed: 12/23/2024]
Abstract
T2DM detection methods are commonly used in teens and adults but are generally unsuitable to unborn fetuses in the context of non-invasive prenatal testing (NIPT). Biophysical and biochemical tests for fetuses are often invasive, carry risks, and have low sensitivity and specificity, with no direct method available to diagnose T2DM in utero. In contrast, cell-free DNA (cfDNA) is known have high sensitivity (93-98 %) and specificity (94-100 %) for cancer detection and fetal genetic disorders (trisomy 21, 8, and 13) making it applicable for fetal epigenetic and genetic analysis, including T2DM early detection. However, no study has explored its use for this purpose. Our review focuses on the potential of IGFBP methylation levels in cfDNA as biomarkers for NIPT of T2DM. Placental global hypomethylation in GDM may predict T2DM during the prenatal period, and a similar pattern potentially be detected in cfDNA. Targeted genes reliable for NIPT, such as IGFBPs are needed because their significant role in T2DM and GDM. Among these, IGFBP-1 and IGFBP-2 have shown potential as predictive genes, exhibiting hypermethylation in placental tissue from GDM cases. This hypermethylation reduces their expression and the formation of the IGF-1-IGFBP complex, leading to increased levels of free IGF-1, which is associated with T2DM in the fetus. Hypermethylation regions have longer fragment sizes in cfDNA, thus in T2DM cases, hypermethylation of IGFBP-1 and IGFBP-2 from fetus results in longer cfDNA fragments. Therefore, analyzing the methylation levels and fragment sizes of IGFBP-1 or IGFBP-2 cfDNA could be a promising biomarker for identifying fetal T2DM risk non-invasively.
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Affiliation(s)
- Audrey Belinda
- Faculty of Biotechnology, University of Surabaya, Surabaya 60292, Indonesia.
| | | | | | - Bhanu Widyadhana
- Faculty of Biotechnology, University of Surabaya, Surabaya 60292, Indonesia.
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Zhang Z, Su J, Xue J, Xiao L, Hong L, Cai G, Gu T. The Research Progress of DNA Methylation in the Development and Function of the Porcine Placenta. Int J Mol Sci 2024; 25:10687. [PMID: 39409016 PMCID: PMC11476760 DOI: 10.3390/ijms251910687] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2024] [Revised: 09/26/2024] [Accepted: 10/03/2024] [Indexed: 10/20/2024] Open
Abstract
The pig is the most widely consumed domestic animal in China, providing over half of the meat supply in food markets. For livestock, a key economic trait is the reproductive performance, which is significantly influenced by placental development. The placenta, a temporary fetal organ, is crucial for establishing maternal-fetal communication and supporting fetal growth throughout pregnancy. DNA methylation is an epigenetic modification that can regulate the gene expression by recruiting proteins involved in gene silencing or preventing transcription factor binding. To enhance our understanding of the molecular mechanisms underlying DNA methylation in porcine placental development, this review summarizes the structure and function of the porcine placenta and the role of DNA methylation in placental development.
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Affiliation(s)
- Zhiyuan Zhang
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China; (Z.Z.); (J.S.); (J.X.); (L.X.); (L.H.); (G.C.)
| | - Jiawei Su
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China; (Z.Z.); (J.S.); (J.X.); (L.X.); (L.H.); (G.C.)
| | - Jiaming Xue
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China; (Z.Z.); (J.S.); (J.X.); (L.X.); (L.H.); (G.C.)
| | - Liyao Xiao
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China; (Z.Z.); (J.S.); (J.X.); (L.X.); (L.H.); (G.C.)
| | - Linjun Hong
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China; (Z.Z.); (J.S.); (J.X.); (L.X.); (L.H.); (G.C.)
| | - Gengyuan Cai
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China; (Z.Z.); (J.S.); (J.X.); (L.X.); (L.H.); (G.C.)
| | - Ting Gu
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China; (Z.Z.); (J.S.); (J.X.); (L.X.); (L.H.); (G.C.)
- Guangdong Provincial Key Laboratory of Agri-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou 510642, China
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Jeddi F, Faghfuri E, Mehranfar S, Soozangar N. The common bisulfite-conversion-based techniques to analyze DNA methylation in human cancers. Cancer Cell Int 2024; 24:240. [PMID: 38982390 PMCID: PMC11234524 DOI: 10.1186/s12935-024-03405-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2024] [Accepted: 06/11/2024] [Indexed: 07/11/2024] Open
Abstract
DNA methylation is an important molecular modification that plays a key role in the expression of cancer genes. Evaluation of epigenetic changes, hypomethylation and hypermethylation, in specific genes are applied for cancer diagnosis. Numerous studies have concentrated on describing DNA methylation patterns as biomarkers for cancer diagnosis monitoring and predicting response to cancer therapy. Various techniques for detecting DNA methylation status in cancers are based on sodium bisulfite treatment. According to the application of these methods in research and clinical studies, they have a number of advantages and disadvantages. The current review highlights sodium bisulfite treatment-based techniques, as well as, the advantages, drawbacks, and applications of these methods in the evaluation of human cancers.
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Affiliation(s)
- Farhad Jeddi
- Zoonoses Research Center, Ardabil University of Medical Sciences, Ardabil, Iran
- Department of Genetics and Pathology, School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran
| | - Elnaz Faghfuri
- Digestive Diseases Research Center, Ardabil University of Medical Sciences, Ardabil, Iran
| | - Sahar Mehranfar
- Department of Genetics and Immunology, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran
| | - Narges Soozangar
- Zoonoses Research Center, Ardabil University of Medical Sciences, Ardabil, Iran.
- Digestive Diseases Research Center, Ardabil University of Medical Sciences, Ardabil, Iran.
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Rendek T, Pos O, Duranova T, Saade R, Budis J, Repiska V, Szemes T. Current Challenges of Methylation-Based Liquid Biopsies in Cancer Diagnostics. Cancers (Basel) 2024; 16:2001. [PMID: 38893121 PMCID: PMC11171112 DOI: 10.3390/cancers16112001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2024] [Revised: 05/13/2024] [Accepted: 05/16/2024] [Indexed: 06/21/2024] Open
Abstract
In current clinical practice, effective cancer testing and screening paradigms are limited to specific types of cancer, exhibiting varying efficiency, acceptance, and adherence. Cell-free DNA (cfDNA) methylation profiling holds promise in providing information about the presence of malignity regardless of its type and location while leveraging blood-based liquid biopsies as a method to obtain analytical samples. However, technical difficulties, costs and challenges resulting from biological variations, tumor heterogeneity, and exogenous factors persist. This method exploits the mechanisms behind cfDNA release but faces issues like fragmentation, low concentrations, and high background noise. This review explores cfDNA methylation's origins, means of detection, and profiling for cancer diagnostics. The critical evaluation of currently available multi-cancer early detection methods (MCEDs) as well as tests targeting single genes, emphasizing their potential and limits to refine strategies for early cancer detection, are explained. The current methodology limitations, workflows, comparisons of clinically approved liquid biopsy-based methylation tests for cancer, their utilization in companion diagnostics as well as the biological limitations of the epigenetics approach are discussed, aiming to help healthcare providers as well as researchers to orient themselves in this increasingly complex and evolving field of diagnostics.
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Affiliation(s)
- Tomas Rendek
- Institute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine, Comenius University, 811 08 Bratislava, Slovakia;
| | - Ondrej Pos
- Geneton Ltd., 841 04 Bratislava, Slovakia; (O.P.); (J.B.); (T.S.)
- Comenius University Science Park, 841 04 Bratislava, Slovakia;
| | | | - Rami Saade
- 2nd Department of Gynaecology and Obstetrics, Faculty of Medicine, Comenius University, 811 08 Bratislava, Slovakia;
| | - Jaroslav Budis
- Geneton Ltd., 841 04 Bratislava, Slovakia; (O.P.); (J.B.); (T.S.)
- Comenius University Science Park, 841 04 Bratislava, Slovakia;
| | - Vanda Repiska
- Institute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine, Comenius University, 811 08 Bratislava, Slovakia;
| | - Tomas Szemes
- Geneton Ltd., 841 04 Bratislava, Slovakia; (O.P.); (J.B.); (T.S.)
- Comenius University Science Park, 841 04 Bratislava, Slovakia;
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Friedemann M, Jandeck C, Tautz L, Gutewort K, von Rein L, Sukocheva O, Fuessel S, Menschikowski M. Blood-Based DNA Methylation Analysis by Multiplexed OBBPA-ddPCR to Verify Indications for Prostate Biopsies in Suspected Prostate Cancer Patients. Cancers (Basel) 2024; 16:1324. [PMID: 38611002 PMCID: PMC11010987 DOI: 10.3390/cancers16071324] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2024] [Revised: 03/26/2024] [Accepted: 03/27/2024] [Indexed: 04/14/2024] Open
Abstract
Current prostate carcinoma (PCa) biomarkers, including total prostate-specific antigen (tPSA), have unsatisfactory diagnostic sensitivity and specificity resulting in overdiagnosis and overtreatment. Previously, we described an optimised bias-based preamplification-digital droplet PCR (OBBPA-ddPCR) technique, which detects tumour DNA in blood-derived cell-free DNA (cfDNA) of cancer patients. The current study investigated the performance of newly developed OBBPA-ddPCR-based biomarkers. Blood plasma samples from healthy individuals (n = 90, controls) and PCa (n = 39) and benign prostatic hyperplasia patients (BPH, n = 40) were analysed. PCa and BPH patients had tPSA values within a diagnostic grey area of 2-15 ng/mL, for whom further diagnostic validation is most crucial. Methylation levels of biomarkers RASSF1A, MIR129-2, NRIP3, and SOX8 were found significantly increased in PCa patients compared to controls. By combining classical PCa risk factors (percentage of free PSA compared to tPSA (QfPSA) and patient's age) with cfDNA-based biomarkers, we developed PCa risk scores with improved sensitivity and specificity compared to established tPSA and QfPSA single-marker analyses. The diagnostic specificity was increased to 70% with 100% sensitivity for clinically significant PCa patients. Thus, prostate biopsies could be avoided for 28 out of 40 BPH patients. In conclusion, the newly developed risk scores may help to confirm the clinical decision and prevent unnecessary prostate biopsy.
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Affiliation(s)
- Markus Friedemann
- Institute of Clinical Chemistry and Laboratory Medicine, Medical Faculty Carl Gustav Carus, TUD Dresden University of Technology, Fetscherstr. 74, 01307 Dresden, Germany; (C.J.); (K.G.); (L.v.R.)
| | - Carsten Jandeck
- Institute of Clinical Chemistry and Laboratory Medicine, Medical Faculty Carl Gustav Carus, TUD Dresden University of Technology, Fetscherstr. 74, 01307 Dresden, Germany; (C.J.); (K.G.); (L.v.R.)
| | - Lars Tautz
- Joint Practice of Urology “Am Blauen Wunder”, Schillerplatz 2, 01309 Dresden, Germany
| | - Katharina Gutewort
- Institute of Clinical Chemistry and Laboratory Medicine, Medical Faculty Carl Gustav Carus, TUD Dresden University of Technology, Fetscherstr. 74, 01307 Dresden, Germany; (C.J.); (K.G.); (L.v.R.)
| | - Lisa von Rein
- Institute of Clinical Chemistry and Laboratory Medicine, Medical Faculty Carl Gustav Carus, TUD Dresden University of Technology, Fetscherstr. 74, 01307 Dresden, Germany; (C.J.); (K.G.); (L.v.R.)
| | - Olga Sukocheva
- Department of Hepatology, Royal Adelaide Hospital, Port Rd., Adelaide, SA 5000, Australia;
| | - Susanne Fuessel
- Clinic of Urology, Carl Gustav Carus University Hospital, TUD Dresden University of Technology, Fetscherstr. 74, 01307 Dresden, Germany;
| | - Mario Menschikowski
- Institute of Clinical Chemistry and Laboratory Medicine, Medical Faculty Carl Gustav Carus, TUD Dresden University of Technology, Fetscherstr. 74, 01307 Dresden, Germany; (C.J.); (K.G.); (L.v.R.)
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Jiang YH, Liu YS, Wei YC, Jhang JF, Kuo HC, Huang HH, Chan MWY, Lin GL, Cheng WC, Lin SC, Wang HJ. Hypermethylation Loci of ZNF671, IRF8, and OTX1 as Potential Urine-Based Predictive Biomarkers for Bladder Cancer. Diagnostics (Basel) 2024; 14:468. [PMID: 38472940 DOI: 10.3390/diagnostics14050468] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2024] [Revised: 02/11/2024] [Accepted: 02/14/2024] [Indexed: 03/14/2024] Open
Abstract
Bladder cancer (BCa) is a significant health issue and poses a healthcare burden on patients, highlighting the importance of an effective detection method. Here, we developed a urine DNA methylation diagnostic panel for distinguishing between BCa and non-BCa. In the discovery stage, an analysis of the TCGA database was conducted to identify BCa-specific DNA hypermethylation markers. In the validation phase, DNA methylation levels of urine samples were measured with real-time quantitative methylation-specific PCR (qMSP). Comparative analysis of the methylation levels between BCa and non-BCa, along with the receiver operating characteristic (ROC) analyses with machine learning algorithms (logistic regression and decision tree methods) were conducted to develop practical diagnostic panels. The performance evaluation of the panel shows that the individual biomarkers of ZNF671, OTX1, and IRF8 achieved AUCs of 0.86, 0.82, and 0.81, respectively, while the combined yielded an AUC of 0.91. The diagnostic panel using the decision tree algorithm attained an accuracy, sensitivity, and specificity of 82.6%, 75.0%, and 90.9%, respectively. Our results show that the urine-based DNA methylation diagnostic panel provides a sensitive and specific method for detecting and stratifying BCa, showing promise as a standard test that could enhance the diagnosis and prognosis of BCa in clinical settings.
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Affiliation(s)
- Yuan-Hong Jiang
- Department of Urology, Hualien Tzu Chi Hospital, Tzu Chi University, Hualien 970374, Taiwan
| | - Yu-Shu Liu
- Guzip Biomarkers Corporation, Hsinchu City 302041, Taiwan
- Phalanx Biotech, Hsinchu City 302041, Taiwan
| | - Yu-Chung Wei
- Graduate Institute of Statistics and Information Science, National Changhua University of Education, Changhua City 500207, Taiwan
| | - Jia-Fong Jhang
- Department of Urology, Hualien Tzu Chi Hospital, Tzu Chi University, Hualien 970374, Taiwan
| | - Hann-Chorng Kuo
- Department of Urology, Hualien Tzu Chi Hospital, Tzu Chi University, Hualien 970374, Taiwan
- Guzip Biomarkers Corporation, Hsinchu City 302041, Taiwan
| | - Hsin-Hui Huang
- Guzip Biomarkers Corporation, Hsinchu City 302041, Taiwan
- Phalanx Biotech, Hsinchu City 302041, Taiwan
| | - Michael W Y Chan
- Department of Biomedical Sciences, National Chung Cheng University, Minhsiung, Chiayi 621301, Taiwan
- Epigenomics and Human Disease Research Center, National Chung Cheng University, Minhsiung, Chiayi 621301, Taiwan
- Center for Innovative Research on Aging Society (CIRAS), National Chung Cheng University, Min-Hsiung, Chiayi 621301, Taiwan
| | - Guan-Ling Lin
- Department of Biomedical Sciences, National Chung Cheng University, Minhsiung, Chiayi 621301, Taiwan
- Epigenomics and Human Disease Research Center, National Chung Cheng University, Minhsiung, Chiayi 621301, Taiwan
- Center for Innovative Research on Aging Society (CIRAS), National Chung Cheng University, Min-Hsiung, Chiayi 621301, Taiwan
| | - Wen-Chi Cheng
- Institute of Medical Sciences, Tzu Chi University, Hualien 970374, Taiwan
| | - Shu-Chuan Lin
- Guzip Biomarkers Corporation, Hsinchu City 302041, Taiwan
- Phalanx Biotech, Hsinchu City 302041, Taiwan
| | - Hung-Jung Wang
- Institute of Medical Sciences, Tzu Chi University, Hualien 970374, Taiwan
- Doctoral Degree Program in Translational Medicine, Tzu Chi University and Academia Sinica, Hualien 97004, Taiwan
- Department of Biomedical Sciences and Engineering, Tzu Chi University, Hualien 970374, Taiwan
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Zhang L, Li J. Unlocking the secrets: the power of methylation-based cfDNA detection of tissue damage in organ systems. Clin Epigenetics 2023; 15:168. [PMID: 37858233 PMCID: PMC10588141 DOI: 10.1186/s13148-023-01585-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2023] [Accepted: 10/11/2023] [Indexed: 10/21/2023] Open
Abstract
BACKGROUND Detecting organ and tissue damage is essential for early diagnosis, treatment decisions, and monitoring disease progression. Methylation-based assays offer a promising approach, as DNA methylation patterns can change in response to tissue damage. These assays have potential applications in early detection, monitoring disease progression, evaluating treatment efficacy, and assessing organ viability for transplantation. cfDNA released into the bloodstream upon tissue or organ injury can serve as a biomarker for damage. The epigenetic state of cfDNA, including DNA methylation patterns, can provide insights into the extent of tissue and organ damage. CONTENT Firstly, this review highlights DNA methylation as an extensively studied epigenetic modification that plays a pivotal role in processes such as cell growth, differentiation, and disease development. It then presents a variety of highly precise 5-mC methylation detection techniques that serve as powerful tools for gaining profound insights into epigenetic alterations linked with tissue damage. Subsequently, the review delves into the mechanisms underlying DNA methylation changes in organ and tissue damage, encompassing inflammation, oxidative stress, and DNA damage repair mechanisms. Next, it addresses the current research status of cfDNA methylation in the detection of specific organ tissues and organ damage. Finally, it provides an overview of the multiple steps involved in identifying specific methylation markers associated with tissue and organ damage for clinical trials. This review will explore the mechanisms and current state of research on cfDNA methylation-based assay detecting organ and tissue damage, the underlying mechanisms, and potential applications in clinical practice.
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Affiliation(s)
- Lijing Zhang
- National Center for Clinical Laboratories, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing Hospital/National Center of Gerontology, No. 1 Dahua Road, Dongdan, Beijing, 100730, People's Republic of China
- Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing Hospital, Beijing, People's Republic of China
- Beijing Engineering Research Center of Laboratory Medicine, Beijing, People's Republic of China
| | - Jinming Li
- National Center for Clinical Laboratories, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing Hospital/National Center of Gerontology, No. 1 Dahua Road, Dongdan, Beijing, 100730, People's Republic of China.
- Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing Hospital, Beijing, People's Republic of China.
- Beijing Engineering Research Center of Laboratory Medicine, Beijing, People's Republic of China.
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Zhang J, Sheng H, Hu C, Li F, Cai B, Ma Y, Wang Y, Ma Y. Effects of DNA Methylation on Gene Expression and Phenotypic Traits in Cattle: A Review. Int J Mol Sci 2023; 24:11882. [PMID: 37569258 PMCID: PMC10419045 DOI: 10.3390/ijms241511882] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2023] [Revised: 07/20/2023] [Accepted: 07/22/2023] [Indexed: 08/13/2023] Open
Abstract
Gene expression in cells is determined by the epigenetic state of chromatin. Therefore, the study of epigenetic changes is very important to understand the regulatory mechanism of genes at the molecular, cellular, tissue and organ levels. DNA methylation is one of the most studied epigenetic modifications, which plays an important role in maintaining genome stability and ensuring normal growth and development. Studies have shown that methylation levels in bovine primordial germ cells, the rearrangement of methylation during embryonic development and abnormal methylation during placental development are all closely related to their reproductive processes. In addition, the application of bovine male sterility and assisted reproductive technology is also related to DNA methylation. This review introduces the principle, development of detection methods and application conditions of DNA methylation, with emphasis on the relationship between DNA methylation dynamics and bovine spermatogenesis, embryonic development, disease resistance and muscle and fat development, in order to provide theoretical basis for the application of DNA methylation in cattle breeding in the future.
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Affiliation(s)
- Junxing Zhang
- Key Laboratory of Ruminant Molecular Cell Breeding of Ningxia Hui Autonomous Region, College of Animal Science and Technology, Ningxia University, Yinchuan 750021, China; (J.Z.); (H.S.); (C.H.); (F.L.); (B.C.); (Y.M.)
- College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
| | - Hui Sheng
- Key Laboratory of Ruminant Molecular Cell Breeding of Ningxia Hui Autonomous Region, College of Animal Science and Technology, Ningxia University, Yinchuan 750021, China; (J.Z.); (H.S.); (C.H.); (F.L.); (B.C.); (Y.M.)
| | - Chunli Hu
- Key Laboratory of Ruminant Molecular Cell Breeding of Ningxia Hui Autonomous Region, College of Animal Science and Technology, Ningxia University, Yinchuan 750021, China; (J.Z.); (H.S.); (C.H.); (F.L.); (B.C.); (Y.M.)
| | - Fen Li
- Key Laboratory of Ruminant Molecular Cell Breeding of Ningxia Hui Autonomous Region, College of Animal Science and Technology, Ningxia University, Yinchuan 750021, China; (J.Z.); (H.S.); (C.H.); (F.L.); (B.C.); (Y.M.)
| | - Bei Cai
- Key Laboratory of Ruminant Molecular Cell Breeding of Ningxia Hui Autonomous Region, College of Animal Science and Technology, Ningxia University, Yinchuan 750021, China; (J.Z.); (H.S.); (C.H.); (F.L.); (B.C.); (Y.M.)
| | - Yanfen Ma
- Key Laboratory of Ruminant Molecular Cell Breeding of Ningxia Hui Autonomous Region, College of Animal Science and Technology, Ningxia University, Yinchuan 750021, China; (J.Z.); (H.S.); (C.H.); (F.L.); (B.C.); (Y.M.)
| | - Yachun Wang
- College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
| | - Yun Ma
- Key Laboratory of Ruminant Molecular Cell Breeding of Ningxia Hui Autonomous Region, College of Animal Science and Technology, Ningxia University, Yinchuan 750021, China; (J.Z.); (H.S.); (C.H.); (F.L.); (B.C.); (Y.M.)
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11
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Oh TJ, Lim E, Bang BR, Lee JJ, Na YG, Shin JH, Lim JS, Song KH, An S. Identification and validation of methylated PENK gene for early detection of bladder cancer using urine DNA. BMC Cancer 2022; 22:1195. [PMID: 36403035 PMCID: PMC9675278 DOI: 10.1186/s12885-022-10275-2] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2021] [Accepted: 11/02/2022] [Indexed: 11/21/2022] Open
Abstract
Background Early detection of bladder cancer (BCa) offers patients a favorable outcome and avoids the need for cystectomy. Development of an accurate and sensitive noninvasive BCa diagnostic test is imperative. DNA methylation is an early epigenetic event in the development of BCa. Certain specific aberrant methylations could serve as useful biomarkers. The aim of this study was to identify methylation biomarkers for early detection of BCa. Methods CpG methylation microarray analysis was conducted on primary tumors with varying stages (T1—T4) and paired nontumor tissues from nine BCa patients. Bisulfite-pyrosequencing was performed to confirm the methylation status of candidate genes in tissues and urine sediments (n = 51). Among them, PENK was selected as a potential candidate and validated using an independent set of 169 urine sediments (55 BCa, 25 benign urologic diseases, 8 other urologic cancers, and 81 healthy controls) with a quantitative methylation-specific real time PCR (mePENK-qMSP). All statistical analyses were performed using MedCalc software version 9.3.2.0. Results CpG methylation microarray analysis and stepwise validation by bisulfite-pyrosequencing for tissues and urine sediments supported aberrant methylation sites of the PENK gene as potential biomarkers for early detection of BCa. Clinical validation of the mePENK-qMSP test using urine sediment-DNA showed a sensitivity of 86.5% (95% CI: 71.2 – 95.5%), a specificity of 92.5% (95% CI: 85.7 – 96.7%), and an area under ROC of 0.920 (95% CI: 0.863 – 0.959) in detecting Ta high-grade and advanced tumor stages (T1-T4) of BCa patients. Sensitivities for Ta low-grade, Ta high-grade, T1 and T2-T4 were 55.6, 83.3, 88.5, and 100%, respectively. Methylation status of PENK was not correlated with sex, age or stage, while it was associated with the tumor grade of BCa. Conclusions In this study, we analyzed the comprehensive patterns of DNA methylation identified that PENK methylation possesses a high potential as a biomarker for urine-based early detection of BCa. Validation of PENK methylation confirms that it could significantly improve the noninvasive detection of BCa. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-022-10275-2.
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Affiliation(s)
- Tae Jeong Oh
- Genomictree, Inc., 44-6 Techno 10-Ro Yuseong-Gu, Daejeon, 34027 Republic of Korea
| | - Eunkyung Lim
- Genomictree, Inc., 44-6 Techno 10-Ro Yuseong-Gu, Daejeon, 34027 Republic of Korea
| | - Bo-Ram Bang
- Promis Diagnostics Inc., 1 Post, Irvine, CA 92618 USA
| | | | - Yong Gil Na
- grid.254230.20000 0001 0722 6377Department of Urology, Chungnam National University College of Medicine, 266 Munhwa-Ro Jung-Gu, Daejeon, 35015 Republic of Korea
| | - Ju Hyun Shin
- grid.254230.20000 0001 0722 6377Department of Urology, Chungnam National University College of Medicine, 266 Munhwa-Ro Jung-Gu, Daejeon, 35015 Republic of Korea
| | - Jae Sung Lim
- grid.254230.20000 0001 0722 6377Department of Urology, Chungnam National University College of Medicine, 266 Munhwa-Ro Jung-Gu, Daejeon, 35015 Republic of Korea
| | - Ki Hak Song
- grid.254230.20000 0001 0722 6377Department of Urology, Chungnam National University College of Medicine, 266 Munhwa-Ro Jung-Gu, Daejeon, 35015 Republic of Korea
| | - Sungwhan An
- Genomictree, Inc., 44-6 Techno 10-Ro Yuseong-Gu, Daejeon, 34027 Republic of Korea ,Promis Diagnostics Inc., 1 Post, Irvine, CA 92618 USA
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12
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Wang LJ, Han Q, Qiu JG, Zhang CY. Cooperative In Situ Assembly of G-Quadruplex DNAzyme Nanowires for One-Step Sensing of CpG Methylation in Human Genomes. NANO LETTERS 2022; 22:347-354. [PMID: 34931851 DOI: 10.1021/acs.nanolett.1c03969] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
CpG methylation is one the most predominant epigenetic modification that has been recognized as a molecular-level biomarker for various human diseases. Taking advantage of methylation-dependent cleavage and encoding flexibility in nucleic acid functions and structures, we demonstrate the cooperative in situ assembly of G-quadruplex DNAzyme nanowires for one-step sensing of CpG methylation in human genomes. This nanodevice displays good specificity and high sensitivity with a limit of detection (LOD) of 0.565 aM in vitro and 1 cell in vivo. It can distinguish 0.001% CpG methylation level from excess unmethylated DNA, quantify different CpG methylation targets from diverse human cancer cells, and even discriminate CpG methylation expressions between lung tumor and precancerous tissues. Importantly, this nanodevice can be performed isothermally in one step within 2 h in a label-free manner without any bisulfite conversion, fluorescence tagging, and PCR amplification process, providing a new platform for genomic methylation-related clinical diagnosis and biomedical research.
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Affiliation(s)
- Li-Juan Wang
- College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, China
- School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China
| | - Qian Han
- College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, China
| | - Jian-Ge Qiu
- Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan 450000, China
| | - Chun-Yang Zhang
- College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, China
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13
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Tost J. Current and Emerging Technologies for the Analysis of the Genome-Wide and Locus-Specific DNA Methylation Patterns. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2022; 1389:395-469. [DOI: 10.1007/978-3-031-11454-0_16] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
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14
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Wang X, Zhou S, Chu C, Yang M, Huo D, Hou C. Dual Methylation-Sensitive Restriction Endonucleases Coupling with an RPA-Assisted CRISPR/Cas13a System (DESCS) for Highly Sensitive Analysis of DNA Methylation and Its Application for Point-of-Care Detection. ACS Sens 2021; 6:2419-2428. [PMID: 34019391 DOI: 10.1021/acssensors.1c00674] [Citation(s) in RCA: 57] [Impact Index Per Article: 14.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
High-performance detection of DNA methylation possesses great significance for the diagnosis and therapy of cancer. Herein, for the first time, we present a digestion strategy based on dual methylation-sensitive restriction endonucleases coupling with a recombinase polymerase amplification (RPA)-assisted CRISPR/Cas13a system (DESCS) for accurate and sensitive determination of site-specific DNA methylation. This dual methylation-sensitive restriction endonuclease system selectively digests the unmethylated target but exhibits no response to methylated DNA. Therefore, the intact methylated DNA target triggers the RPA reaction for rapid signal amplification. In contrast, the digested unmethylated target initiates no RPA reaction. RPA products with a T7 promoter can execute the T7 transcription in the presence of T7 RNA polymerase to generate a large number of single-stranded RNA (ssRNA). This ssRNA can be recognized by CRISPR/Cas13a to induce the ssRNase activity of Cas13a, showing the indiscriminate cleavage of the collateral FQ reporter to release the fluorescence signal. With such a design, by combining the unique features of dual methylation-sensitive restriction endonucleases with RPA-assisted CRISPR/Cas13a, the DESCS system not only presents the rapid and powerful signal amplification for the determination of methylated DNA with ultrahigh sensitivity but also effectively eliminates the false positive influences from incomplete digestion of the unmethylated target. More importantly, 0.01% methylation level can be effectively distinguished with the existence of excess unmethylated DNA. In addition, the DESCS assay is integrated into the lateral flow biosensor (LFB) for the point-of-care determination of DNA methylation. In view of the superiorities in high sensitivity, outstanding selectivity, and ease of operation, the DESCS system will provide a reliable assay for site-specific analysis of methylation.
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Affiliation(s)
- Xianfeng Wang
- Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing 400044, P. R. China
| | - Shiying Zhou
- Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing 400044, P. R. China
| | - Chengxiang Chu
- Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing 400044, P. R. China
| | - Mei Yang
- Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing 400044, P. R. China
| | - Danqun Huo
- Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing 400044, P. R. China
- Chongqing Key Laboratory of Bio-perception & Intelligent Information Processing, School of Microelectronics and Communication Engineering, Chongqing University, Chongqing 400044, P. R. China
| | - Changjun Hou
- Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing 400044, P. R. China
- Chongqing Key Laboratory of Bio-perception & Intelligent Information Processing, School of Microelectronics and Communication Engineering, Chongqing University, Chongqing 400044, P. R. China
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15
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Goh SK, Cox DRA, Wong BKL, Musafer A, Witkowski T, Do H, Muralidharan V, Dobrovic A. A Synthetic DNA Construct to Evaluate the Recovery Efficiency of Cell-Free DNA Extraction and Bisulfite Modification. Clin Chem 2021; 67:1201-1209. [PMID: 34151944 DOI: 10.1093/clinchem/hvab095] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2020] [Accepted: 04/20/2021] [Indexed: 12/18/2022]
Abstract
BACKGROUND Despite improvements in the genetic and epigenetic analysis of cell-free DNA (cfDNA), there has been limited focus on assessing the preanalytical variables of recovery efficiency following cfDNA extraction and bisulfite modification. Quantification of recovery efficiency after these steps can facilitate quality assurance and improve reliability when comparing serial samples. METHODS We developed an exogenous DNA Construct to Evaluate the Recovery Efficiency of cfDNA extraction and BISulfite modification (CEREBIS) after cfDNA extraction and/or subsequent bisulfite modification from plasma. The strategic placement of cytosine bases in the 180 bp CEREBIS enabled PCR amplification of the construct by a single primer set both after plasma DNA extraction and following subsequent bisulfite modification. RESULTS Plasma samples derived from 8 organ transplant donors and 6 serial plasma samples derived from a liver transplant recipient were spiked with a known number of copies of CEREBIS. Recovery of CEREBIS after cfDNA extraction and bisulfite modification was quantified with high analytical accuracy by droplet digital PCR. The use of CEREBIS and quantification of its recovery was useful in identifying problematic extractions. Furthermore, its use was shown to be invaluable towards improving the reliability of the analysis of serial samples. CONCLUSIONS CEREBIS can be used as a spike-in control to address the preanalytical variable of recovery efficiency both after cfDNA extraction from plasma and following bisulfite modification. Our approach can be readily implemented and its application may have significant benefits, especially in settings where longitudinal quantification of cfDNA for disease monitoring is necessary.
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Affiliation(s)
- Su Kah Goh
- Department of Surgery-Austin Precinct, The University of Melbourne, Melbourne, Victoria, Australia.,Department of Surgery, Austin Hospital, HPB & Transplant Unit, Melbourne, Victoria, Australia.,Department of Surgery-Austin Precinct, Translational Genomics and Epigenomics Laboratory, The University of Melbourne, Melbourne, Victoria, Australia
| | - Daniel R A Cox
- Department of Surgery-Austin Precinct, The University of Melbourne, Melbourne, Victoria, Australia.,Department of Surgery, Austin Hospital, HPB & Transplant Unit, Melbourne, Victoria, Australia.,Department of Surgery-Austin Precinct, Translational Genomics and Epigenomics Laboratory, The University of Melbourne, Melbourne, Victoria, Australia
| | - Boris Ka Leong Wong
- Department of Surgery-Austin Precinct, Translational Genomics and Epigenomics Laboratory, The University of Melbourne, Melbourne, Victoria, Australia.,School of Cancer Medicine, La Trobe University, Bundoora, Victoria, Australia.,Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, Australia
| | - Ashan Musafer
- Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, Australia
| | - Tom Witkowski
- Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, Australia
| | - Hongdo Do
- School of Cancer Medicine, La Trobe University, Bundoora, Victoria, Australia.,The University of Melbourne, Parkville Precinct, Victoria, Australia.,Pathology Department, St Vincent's Hospital Melbourne, Fitzroy, Victoria, Australia
| | - Vijayaragavan Muralidharan
- Department of Surgery-Austin Precinct, The University of Melbourne, Melbourne, Victoria, Australia.,Department of Surgery, Austin Hospital, HPB & Transplant Unit, Melbourne, Victoria, Australia
| | - Alexander Dobrovic
- Department of Surgery-Austin Precinct, The University of Melbourne, Melbourne, Victoria, Australia.,Department of Surgery-Austin Precinct, Translational Genomics and Epigenomics Laboratory, The University of Melbourne, Melbourne, Victoria, Australia.,School of Cancer Medicine, La Trobe University, Bundoora, Victoria, Australia.,Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, Australia
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16
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Jakobsen T, Dahl M, Dimopoulos K, Grønbæk K, Kjems J, Kristensen LS. Genome-Wide Circular RNA Expression Patterns Reflect Resistance to Immunomodulatory Drugs in Multiple Myeloma Cells. Cancers (Basel) 2021; 13:cancers13030365. [PMID: 33498476 PMCID: PMC7930955 DOI: 10.3390/cancers13030365] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2020] [Revised: 01/09/2021] [Accepted: 01/16/2021] [Indexed: 12/15/2022] Open
Abstract
Simple Summary Multiple myeloma (MM) constitutes the second most common hematological malignancy and is caused by aberrant plasma cell proliferation in the bone marrow. While recent improvements in the treatment of MM has been observed using immunomodulatory drugs (IMiDs), patients often relapse due to acquired drug resistance and no cure for the disease is currently available. In this report, we profile circular RNA (circRNA) expression patterns in cultured MM cells being sensitive to IMiDs and their resistant counterparts. CircRNAs constitute a large class of non-coding RNA molecules with emerging roles in cancer development and progression, but have not previously been explored in this context. We found that global circRNA expression patterns reflect IMiD sensitivity, but the most downregulated circRNA in IMiD resistant MM cells did not seem to be a direct driver of IMiD resistance. Future studies should investigate other circRNA candidates identified here in the context of IMiD resistance. Abstract Immunomodulatory drugs (IMiDs), such as lenalidomide and pomalidomide, may induce significant remissions in multiple myeloma (MM) patients, but relapses are frequently observed and the underlying molecular mechanisms for this are not completely understood. Circular RNAs (circRNAs) constitute an emerging class of non-coding RNAs with important roles in cancer. Here, we profiled genome-wide expression patterns of circRNAs in IMiD-sensitive MM cells and their resistant counterparts as well as in IMiD-resistant cells treated with specific epigenetic drugs alone or in combination. We found that genome-wide circRNA expression patterns reflect IMiD sensitivity and ciRS-7 (also known as CDR1as) was the most downregulated circRNA upon acquired resistance. The depletion of ciRS-7 correlated with increased methylation levels of the promoter CpG island of its host gene, LINC00632. Expression of LINC00632 and ciRS-7 was partly restored by treatment with a combination of an EZH2 inhibitor (EPZ-6438) and a DNA methyl transferase inhibitor (5-azacytidine), which also restores the IMiD sensitivity of the cells. However, knockdown of ciRS-7 did not affect IMiD sensitivity and we found that ciRS-7 also becomes epigenetically silenced after prolonged cell culture without drug-exposure. In conclusion, we found that genome-wide circRNA expression patterns reflect IMiD sensitivity in an in vitro model of acquired resistance.
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Affiliation(s)
- Theresa Jakobsen
- Department of Biomedicine, Aarhus University, Høegh-Guldbergs Gade 10, DK-8000 Aarhus, Denmark;
| | - Mette Dahl
- Department of Hematology, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen, Denmark; (M.D.); (K.D.); (K.G.)
- Biotech Research and Innovation Centre, BRIC, Copenhagen University, Ole Maaløes Vej 5, DK-2200 Copenhagen, Denmark
| | - Konstantinos Dimopoulos
- Department of Hematology, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen, Denmark; (M.D.); (K.D.); (K.G.)
- Biotech Research and Innovation Centre, BRIC, Copenhagen University, Ole Maaløes Vej 5, DK-2200 Copenhagen, Denmark
| | - Kirsten Grønbæk
- Department of Hematology, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen, Denmark; (M.D.); (K.D.); (K.G.)
- Biotech Research and Innovation Centre, BRIC, Copenhagen University, Ole Maaløes Vej 5, DK-2200 Copenhagen, Denmark
| | - Jørgen Kjems
- Department of Molecular Biology and Genetics (MBG), Aarhus University, C.F. Møllers Allé 3, DK-8000 Aarhus, Denmark;
- Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, DK-8000 Aarhus, Denmark
| | - Lasse Sommer Kristensen
- Department of Biomedicine, Aarhus University, Høegh-Guldbergs Gade 10, DK-8000 Aarhus, Denmark;
- Correspondence:
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17
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Xu T, Fan X, Zhao M, Wu M, Li H, Ji B, Zhu X, Li L, Ding H, Sun M, Xu Z, Gao Q. DNA Methylation-Reprogrammed Ang II (Angiotensin II) Type 1 Receptor-Early Growth Response Gene 1-Protein Kinase C ε Axis Underlies Vascular Hypercontractility in Antenatal Hypoxic Offspring. Hypertension 2020; 77:491-506. [PMID: 33342239 DOI: 10.1161/hypertensionaha.120.16247] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
As the most common clinical stress during mid and late pregnancy, antenatal hypoxia has profound adverse effects on individual's vascular health later in life, but the underlying mechanisms are still not understood. The purpose of this study was to reveal the mechanisms of the acquired vascular dysfunction in offspring imposed by antenatal hypoxia. Pregnant rats were housed in a normoxic or hypoxic (10.5% oxygen) chamber from gestation day 10 to 21. Male offspring were euthanized at gestational day 21 (fetus) or postnatal 16 weeks old (adult offspring). Mesenteric arteries were collected for examining Ang II (angiotensin II)-mediated vascular contractility, gene expression, and promoter methylation. Antenatal hypoxia increased vascular sensitivity to Ang II, which was resulted by an upregulated AT1R (angiotensin II type 1 receptor). The increased AT1R was correlated with a hypomethylation-mediated activated transcription of Agtr1a (alpha subtype of AT1R). In addition, we presented evidences that there was an AT1R-Egr1 (early growth response gene 1)-PKCε (ε isoform of protein kinase C) axis in vasculature; AT1R could modulate PKCε expression via upregulating Egr1; Egr1 mediated transcription activation of PKCε via Egr1 binding sites in PKCε gene promoter. Overall, antenatal hypoxia activated AT1R-Egr1-PKCε axis in vasculature, eventually predisposed offspring to vascular hypercontractility. This is the first description that antenatal hypoxia resulted in vascular adverse outcomes in postnatal offspring, was strongly associated with reprogrammed gene expression via a DNA methylation-mediated epigenetic mechanism, advancing understanding toward the influence of adverse antenatal factors in early life on long-term vascular health.
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Affiliation(s)
- Ting Xu
- From the Institute for Fetology (T.X., M.Z., H.L., B.J., X.Z., L.L., M.S., Z.X., Q.G.), First Hospital of Soochow University, Suzhou, China
| | - Xiaorong Fan
- Institute of Reproductive Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China (X.F.)
| | - Meng Zhao
- From the Institute for Fetology (T.X., M.Z., H.L., B.J., X.Z., L.L., M.S., Z.X., Q.G.), First Hospital of Soochow University, Suzhou, China
| | - Meng Wu
- Institutes of Biological and Medical Sciences, Soochow University Medical School, Suzhou, China (M.W.)
| | - Huan Li
- From the Institute for Fetology (T.X., M.Z., H.L., B.J., X.Z., L.L., M.S., Z.X., Q.G.), First Hospital of Soochow University, Suzhou, China
| | - Bingyu Ji
- From the Institute for Fetology (T.X., M.Z., H.L., B.J., X.Z., L.L., M.S., Z.X., Q.G.), First Hospital of Soochow University, Suzhou, China
| | - Xiaolin Zhu
- From the Institute for Fetology (T.X., M.Z., H.L., B.J., X.Z., L.L., M.S., Z.X., Q.G.), First Hospital of Soochow University, Suzhou, China
| | - Lingjun Li
- From the Institute for Fetology (T.X., M.Z., H.L., B.J., X.Z., L.L., M.S., Z.X., Q.G.), First Hospital of Soochow University, Suzhou, China
| | - Hongmei Ding
- Department of Obstetrics and Gynecology (H.D.), First Hospital of Soochow University, Suzhou, China
| | - Miao Sun
- From the Institute for Fetology (T.X., M.Z., H.L., B.J., X.Z., L.L., M.S., Z.X., Q.G.), First Hospital of Soochow University, Suzhou, China
| | - Zhice Xu
- From the Institute for Fetology (T.X., M.Z., H.L., B.J., X.Z., L.L., M.S., Z.X., Q.G.), First Hospital of Soochow University, Suzhou, China
| | - Qinqin Gao
- From the Institute for Fetology (T.X., M.Z., H.L., B.J., X.Z., L.L., M.S., Z.X., Q.G.), First Hospital of Soochow University, Suzhou, China
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18
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You T, Song K, Guo W, Fu Y, Wang K, Zheng H, Yang J, Jin L, Qi L, Guo Z, Zhao W. A Qualitative Transcriptional Signature for Predicting CpG Island Methylator Phenotype Status of the Right-Sided Colon Cancer. Front Genet 2020; 11:971. [PMID: 33193579 PMCID: PMC7658404 DOI: 10.3389/fgene.2020.00971] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2020] [Accepted: 07/31/2020] [Indexed: 12/24/2022] Open
Abstract
A part of colorectal cancer which is characterized by simultaneous numerous hypermethylation CpG islands sites is defined as CpG island methylator phenotype (CIMP) status. Stage II and III CIMP−positive (CIMP+) right-sided colon cancer (RCC) patients have a better prognosis than CIMP−negative (CIMP−) RCC treated with surgery alone. However, there is no gold standard available in defining CIMP status. In this work, we selected the gene pairs whose relative expression orderings (REOs) were associated with the CIMP status, to develop a qualitative transcriptional signature to individually predict CIMP status for stage II and III RCC. Based on the REOs of gene pairs, a signature composed of 19 gene pairs was developed to predict the CIMP status of RCC through a feature selection process. A sample is predicted as CIMP+ when the gene expression orderings of at least 12 gene pairs vote for CIMP+; otherwise the CIMP−. The difference of prognosis between the predicted CIMP+ and CIMP− groups was more significantly different than the original CIMP status groups. There were more differential methylation and expression characteristics between the two predicted groups. The hierarchical clustering analysis showed that the signature could perform better for predicting CIMP status of RCC than current methods. In conclusion, the qualitative transcriptional signature for classifying CIMP status at the individualized level can predict outcome and guide therapy for RCC patients.
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Affiliation(s)
- Tianyi You
- Department of Systems Biology, College of Bioinformatics Science and Technology, Harbin Medical University, Harbin, China
| | - Kai Song
- Department of Systems Biology, College of Bioinformatics Science and Technology, Harbin Medical University, Harbin, China
| | - Wenbing Guo
- Department of Systems Biology, College of Bioinformatics Science and Technology, Harbin Medical University, Harbin, China
| | - Yelin Fu
- Department of Systems Biology, College of Bioinformatics Science and Technology, Harbin Medical University, Harbin, China
| | - Kai Wang
- Department of Systems Biology, College of Bioinformatics Science and Technology, Harbin Medical University, Harbin, China
| | - Hailong Zheng
- Department of Systems Biology, College of Bioinformatics Science and Technology, Harbin Medical University, Harbin, China
| | - Jing Yang
- Department of Systems Biology, College of Bioinformatics Science and Technology, Harbin Medical University, Harbin, China
| | - Liangliang Jin
- Department of Systems Biology, College of Bioinformatics Science and Technology, Harbin Medical University, Harbin, China
| | - Lishuang Qi
- Department of Systems Biology, College of Bioinformatics Science and Technology, Harbin Medical University, Harbin, China
| | - Zheng Guo
- Department of Systems Biology, College of Bioinformatics Science and Technology, Harbin Medical University, Harbin, China.,Department of Bioinformatics, Key Laboratory of Ministry of Education for Gastrointestinal Cancer, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China.,Fujian Provincial Key Laboratory on Hematology, Fujian Medical University, Fuzhou, China
| | - Wenyuan Zhao
- Department of Systems Biology, College of Bioinformatics Science and Technology, Harbin Medical University, Harbin, China
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19
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Miller BF, Pisanic Ii TR, Margolin G, Petrykowska HM, Athamanolap P, Goncearenco A, Osei-Tutu A, Annunziata CM, Wang TH, Elnitski L. Leveraging locus-specific epigenetic heterogeneity to improve the performance of blood-based DNA methylation biomarkers. Clin Epigenetics 2020; 12:154. [PMID: 33081832 PMCID: PMC7574234 DOI: 10.1186/s13148-020-00939-w] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2019] [Accepted: 09/21/2020] [Indexed: 12/20/2022] Open
Abstract
BACKGROUND Variation in intercellular methylation patterns can complicate the use of methylation biomarkers for clinical diagnostic applications such as blood-based cancer testing. Here, we describe development and validation of a methylation density binary classification method called EpiClass (available for download at https://github.com/Elnitskilab/EpiClass ) that can be used to predict and optimize the performance of methylation biomarkers, particularly in challenging, heterogeneous samples such as liquid biopsies. This approach is based upon leveraging statistical differences in single-molecule sample methylation density distributions to identify ideal thresholds for sample classification. RESULTS We developed and tested the classifier using reduced representation bisulfite sequencing (RRBS) data derived from ovarian carcinoma tissue DNA and controls. We used these data to perform in silico simulations using methylation density profiles from individual epiallelic copies of ZNF154, a genomic locus known to be recurrently methylated in numerous cancer types. From these profiles, we predicted the performance of the classifier in liquid biopsies for the detection of epithelial ovarian carcinomas (EOC). In silico analysis indicated that EpiClass could be leveraged to better identify cancer-positive liquid biopsy samples by implementing precise thresholds with respect to methylation density profiles derived from circulating cell-free DNA (cfDNA) analysis. These predictions were confirmed experimentally using DREAMing to perform digital methylation density analysis on a cohort of low volume (1-ml) plasma samples obtained from 26 EOC-positive and 41 cancer-free women. EpiClass performance was then validated in an independent cohort of 24 plasma specimens, derived from a longitudinal study of 8 EOC-positive women, and 12 plasma specimens derived from 12 healthy women, respectively, attaining a sensitivity/specificity of 91.7%/100.0%. Direct comparison of CA-125 measurements with EpiClass demonstrated that EpiClass was able to better identify EOC-positive women than standard CA-125 assessment. Finally, we used independent whole genome bisulfite sequencing (WGBS) datasets to demonstrate that EpiClass can also identify other cancer types as well or better than alternative methylation-based classifiers. CONCLUSIONS Our results indicate that assessment of intramolecular methylation density distributions calculated from cfDNA facilitates the use of methylation biomarkers for diagnostic applications. Furthermore, we demonstrated that EpiClass analysis of ZNF154 methylation was able to outperform CA-125 in the detection of etiologically diverse ovarian carcinomas, indicating broad utility of ZNF154 for use as a biomarker of ovarian cancer.
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Affiliation(s)
- Brendan F Miller
- Translational Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Thomas R Pisanic Ii
- Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD, 21218, USA.
| | - Gennady Margolin
- Translational Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Hanna M Petrykowska
- Translational Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Pornpat Athamanolap
- Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, 21218, USA
| | - Alexander Goncearenco
- Translational Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Akosua Osei-Tutu
- Women's Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Christina M Annunziata
- Women's Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Tza-Huei Wang
- Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD, 21218, USA
- Department of Mechanical Engineering, Johns Hopkins University, Baltimore, MD, 21218, USA
- Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, 21218, USA
| | - Laura Elnitski
- Translational Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
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Yu H, Pan R, Gao T, Wu D, Ying J, Duan S. FANCF hypomethylation is associated with colorectal cancer in Han Chinese. TURKISH JOURNAL OF GASTROENTEROLOGY 2020; 31:558-565. [PMID: 32915143 DOI: 10.5152/tjg.2020.19394] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
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.
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Affiliation(s)
- Hang Yu
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang, China
| | - Ranran Pan
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang, China
| | - Tong Gao
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang, China
| | - Dongping Wu
- Department of Medical Oncology, Shaoxing People's Hospital, Shaoxing Hospital of Zhejiang University, Zhejiang, China
| | - Jieer Ying
- Department of Medical Oncology, Zhejiang Cancer Hospital, Hangzhou, Zhejiang, China
| | - Shiwei Duan
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang, China
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Husseiny MI, Fahmy A, Du W, Gu A, Garcia P, Ferreri K, Kandeel F. Development of Quantitative Methylation-Specific Droplet Digital PCR (ddMSP) for Assessment of Natural Tregs. Front Genet 2020; 11:300. [PMID: 32318096 PMCID: PMC7154152 DOI: 10.3389/fgene.2020.00300] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2019] [Accepted: 03/13/2020] [Indexed: 11/13/2022] Open
Abstract
Regulatory T cells (Tregs) suppress immune responses in vivo in an antigen-specific manner. Of clinical relevance, Tregs can be isolated and expanded in vitro while maintaining immunoregulatory function. Tregs are classified as CD4+CD25highCD127low FOXP3+ cells. Demethylation of the Treg-specific demethylation region (TSDR) of FOXP3 is found in natural Tregs (nTregs). We report a method for the characterization of the differential methylation pattern of the FOXP3 TSDR in patient-derived and expanded nTregs. Human TSDR sequences from nTregs (unmethylated sequence) and pancreatic (methylated sequence) cells were amplified and cloned into plasmids. A droplet digital TaqMan probe-based qPCR (ddPCR) assay using methylation-specific primers and probes was employed to quantify unmethylated and methylated sequences. The methylation-specific droplet digital PCR (ddMSP) assay was specific and selective for unmethylated DNA in mixtures with methylated DNA in the range of 5000 copies/μL to less than 1 copy/μL (R 2 = 0.99) even in the presence of non-selective gDNAs. CD4+CD25highCD127lowFOXP3+ human nTregs, in the presence of Dynabeads or activators, were expanded for 21 days. There was a decrease in the unmethylated ratio of Tregs after expansion with essentially the same ratio at days 10, 14, and 17. However, the activator expanded group showed a significant decrease in unmethylated targets at day 21. The suppression activity of activator-expanded nTregs at day 21 was decreased compared to cells expanded with Dynabeads. These data suggest that the ddMSP can quantitatively monitor nTreg expansion in vitro. These data also indicate that the assay is sensitive and specific at differentiating nTregs from other cells and may be useful for rapid screening of nTregs in clinical protocols.
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Affiliation(s)
- Mohamed I Husseiny
- Department of Translational Research & Cellular Therapeutics, Diabetes & Metabolism Research Institute, Beckman Research Institute of City of Hope, Duarte, CA, United States.,Faculty of Pharmacy, Zagazig University, Zagazig, Egypt
| | - Ahmed Fahmy
- East Lancashire Hospitals NHS Trust, Blackburn, United Kingdom
| | - Weiting Du
- Department of Translational Research & Cellular Therapeutics, Diabetes & Metabolism Research Institute, Beckman Research Institute of City of Hope, Duarte, CA, United States
| | - Angel Gu
- Department of Translational Research & Cellular Therapeutics, Diabetes & Metabolism Research Institute, Beckman Research Institute of City of Hope, Duarte, CA, United States
| | - Pablo Garcia
- Department of Translational Research & Cellular Therapeutics, Diabetes & Metabolism Research Institute, Beckman Research Institute of City of Hope, Duarte, CA, United States
| | - Kevin Ferreri
- Department of Translational Research & Cellular Therapeutics, Diabetes & Metabolism Research Institute, Beckman Research Institute of City of Hope, Duarte, CA, United States
| | - Fouad Kandeel
- Department of Translational Research & Cellular Therapeutics, Diabetes & Metabolism Research Institute, Beckman Research Institute of City of Hope, Duarte, CA, United States
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Hypermethylation of tumor necrosis factor decoy receptor gene in non-small cell lung cancer. Oncol Lett 2020; 20:155-164. [PMID: 32565943 PMCID: PMC7286129 DOI: 10.3892/ol.2020.11565] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2019] [Accepted: 03/06/2020] [Indexed: 01/16/2023] Open
Abstract
Abnormal methylation of the TNFRSF10C and TNFRSF10D genes has been observed in numerous types of cancer; however, no studies have investigated the methylation of these genes in non-small cell lung cancer (NSCLC). The aim of the present study was to investigate the association between TNFRSF10C and TNFRSF10D methylation and NSCLC. Methylation levels of 44 pairs of NSCLC tumor tissues and distant non-tumor tissues were analyzed using quantitative methylation specific PCR and methylation reference percentage values (PMR). The methylation levels of the TNFRSF10C gene in NSCLC tumor tissue samples were significantly higher compared with those in the distant non-tumor tissues (median PMR, 2.73% vs. 0.75%; P=0.013). Subgroup analysis demonstrated that the methylation levels of TNFRSF10C in tumor tissues from male patients were significantly higher compared with those in distant non-tumor tissues (median PMR, 2.73% vs. 0.75%; P=0.041). The levels of TNFRSF10C methylation were also higher in the tumor tissues of patients who were non-smokers compared with their distant non-tumor tissues (median PMR, 2.50% vs. 0.63%; P=0.013). TNFRSF10C methylation levels were higher in the tumor tissues from male patients compared with those from female patients (median PMR, 2.50% vs. 0.63%; P=0.031). However, no significant differences in the methylation levels of the TNFRSF10D gene were observed between the sexes. Using the cBioPortal and The Cancer Genome Atlas lung cancer data, it was demonstrated that TNFRSF10C methylation levels were inversely correlated with TNFRSF10C mRNA expression levels (r=-0.379; P=0.008). In addition, demethylation of lung cancer cell lines A549 and NCI-H1299 using 5'-aza-deoxycytidine further confirmed that TNFRSF10C hypomethylation was associated with significant upregulation of TNFRSF10C mRNA expression levels [A549 fold-change (FC)=8; P=1.0×10-4; NCI-H1299 FC=3.163; P=1.143×10-5]. A dual luciferase reporter gene assay was also performed with the insert of TNFRSF10C promoter region, and the results revealed that the TNFRSF10C gene fragment significantly enhanced the transcriptional activity of the reporter gene compared with that in the control group (FC=1.570; P=0.032). Overall, the results of the present study demonstrated that hypermethylation of TNFRSF10C was associated with NSCLC.
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23
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Xu L, Zhou C, Pan R, Tang J, Wang J, Li B, Huang T, Duan S, Xu C. PTPN11 hypomethylation is associated with gastric cancer progression. Oncol Lett 2020; 19:1693-1700. [PMID: 32194661 PMCID: PMC7039138 DOI: 10.3892/ol.2020.11250] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2018] [Accepted: 10/14/2019] [Indexed: 11/06/2022] Open
Abstract
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.
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Affiliation(s)
- Lele Xu
- Department of Gastroenterology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215200, P.R. China
| | - Cong Zhou
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Ranran Pan
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Junjian Tang
- Department of Vascular Surgery, Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu 214000, P.R. China
| | - Jinzhi Wang
- Department of Cell Biology, School of Medicine, Soochow University, Suzhou, Jiangsu 215007, P.R. China
| | - Bin Li
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Tianyi Huang
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Shiwei Duan
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Chunfang Xu
- Department of Gastroenterology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215200, P.R. China
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Chen D, Wang Y, Mo M, Zhang J, Zhang Y, Xu Y, Liu SY, Chen J, Ma Y, Zhang L, Dai Z, Cai C, Zou X. Polymerization retardation isothermal amplification (PRIA): a strategy enables sensitively quantify genome-wide 5-methylcytosine oxides rapidly on handy instruments with nanoscale sample input. Nucleic Acids Res 2019; 47:e119. [PMID: 31418020 PMCID: PMC6821303 DOI: 10.1093/nar/gkz704] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2019] [Revised: 06/25/2019] [Accepted: 08/01/2019] [Indexed: 12/22/2022] Open
Abstract
The current methods for quantifying genome-wide 5-methylcytosine (5mC) oxides are still scarce, mostly restricted with two limitations: assay sensitivity is seriously compromised with cost, assay time and sample input; epigenetic information is irreproducible during polymerase chain reaction (PCR) amplification without bisulfite pretreatment. Here, we propose a novel Polymerization Retardation Isothermal Amplification (PRIA) strategy to directly amplify the minute differences between epigenetic bases and others by arranging DNA polymerase to repetitively pass large electron-withdrawing groups tagged 5mC-oxides. We demonstrate that low abundant 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) in genomic DNA can be accurately quantified within 10 h with 100 ng sample input on a laboratory real-time quantitative PCR instrument, and even multiple samples can be analyzed simultaneously in microplates. The global levels of 5hmC and 5fC in mouse and human brain tissues, rat hippocampal neuronal tissue, mouse kidney tissue and mouse embryonic stem cells were quantified and the observations not only confirm the widespread presence of 5hmC and 5fC but also indicate their significant variation in different tissues and cells. The strategy is easily performed in almost all research and medical laboratories, and would provide the potential capability to other candidate modifications in nucleotides.
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Affiliation(s)
- Danping Chen
- School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, China
| | - Yang Wang
- School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, China
| | - Mingming Mo
- Guangdong Key Laboratory for the Research and Development of Natural Drugs, Guangdong Medical College, Zhanjiang, Guangdong 524023, China
| | - Junjie Zhang
- Guangdong Key Laboratory for the Research and Development of Natural Drugs, Guangdong Medical College, Zhanjiang, Guangdong 524023, China
| | - Yanfei Zhang
- School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, China
| | - Yuzhi Xu
- School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, China
| | - Si-Yang Liu
- School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, China
| | - Jun Chen
- School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, China
| | - Yingjun Ma
- School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, China
| | - Li Zhang
- School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, China
| | - Zong Dai
- School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, China
| | - Chun Cai
- Guangdong Key Laboratory for the Research and Development of Natural Drugs, Guangdong Medical College, Zhanjiang, Guangdong 524023, China
| | - Xiaoyong Zou
- School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, China
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25
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Thakur GK, Sharma T, Krishna Latha T, Banerjee BD, Shah HK, Guleria K. High Resolution Based Quantitative Determination of Methylation Status of CDH1 and VIM Gene in Epithelial Ovarian Cancer. Asian Pac J Cancer Prev 2019; 20:2923-2928. [PMID: 31653136 PMCID: PMC6982649 DOI: 10.31557/apjcp.2019.20.10.2923] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2019] [Indexed: 11/25/2022] Open
Abstract
BACKGROUND DNA promoter methylation is widely explored epigenetic phenomena, known to effect gene expression and further perturbation in cellular homeostasis. Myriad of studies have leveraged promoter methylation and its potential as biomarker for various types of cancer. Aim of present study is to investigate promoter methylation of CDH1 and VIM gene and etiology of epithelial ovarian cancer (EOC). METHODS Most of previous studies were qualitative; we have quantitatively assessed methylation levels in 50 EOC cases and control each through high recognition melt (HRM) technique. RESULTS At 10 % cutoff for CDH1 94% of EOC cases were found to be methylated with mean methylation of 45±13.8, whereas for control mean methylation was found to be 7.3±3.7 amongst 16 % methylation positive control samples. For VIM methylation was detected in 96% of cases with mean of 50.44±11.7 in EOC and in 12% methylation positive samples for control mean methylation was 6.24±4.3. CONCLUSION In short HRM based DNA methylation can serve as a robust and sensitive diagnostic method for promoter methylation detection and as a biomarker for early epithelial ovarian cancer detection.
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Affiliation(s)
- Gaurav Kr Thakur
- Environmental and Molecular Biology Laboratory, Department of Biochemistry, University College of Medical Sciences (Delhi University) and GTB Hospital, Dilshad Garden, Delhi, India
| | - Tusha Sharma
- Environmental and Molecular Biology Laboratory, Department of Biochemistry, University College of Medical Sciences (Delhi University) and GTB Hospital, Dilshad Garden, Delhi, India
| | - T Krishna Latha
- Environmental and Molecular Biology Laboratory, Department of Biochemistry, University College of Medical Sciences (Delhi University) and GTB Hospital, Dilshad Garden, Delhi, India
| | - B D Banerjee
- Environmental and Molecular Biology Laboratory, Department of Biochemistry, University College of Medical Sciences (Delhi University) and GTB Hospital, Dilshad Garden, Delhi, India
| | - Harendra Kr Shah
- Environmental and Molecular Biology Laboratory, Department of Biochemistry, University College of Medical Sciences (Delhi University) and GTB Hospital, Dilshad Garden, Delhi, India
| | - Kiran Guleria
- Department of Obst and Gynae, University College of Medical Sciences (Delhi University) and GTB Hospital, Dilshad Garden, Delhi, India
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Ren J, Cui JP, Luo M, Liu H, Hao P, Wang X, Zhang GH. The prevalence and persistence of aberrant promoter DNA methylation in benzene-exposed Chinese workers. PLoS One 2019; 14:e0220500. [PMID: 31381583 PMCID: PMC6681966 DOI: 10.1371/journal.pone.0220500] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2018] [Accepted: 07/17/2019] [Indexed: 12/13/2022] Open
Abstract
Aberrant DNA methylation patterns are common in cancers and environmental pollutant exposed subjects. Up to date, few studies have examined the aberrant DNA methylation patterns in benzene exposed workers. We recruited 141 benzene-exposed workers, including 83 benzene-exposed workers from a shoe factory in Wenzhou and 58 workers from a painting workshop in Wuhu, 35 workers in Wuhu were followed from 2009 to 2013, and 48 indoor workers as controls from Wenzhou. We used high-resolution melting (HRM) to quantitate human samples of DNA methylation in long interspersed nuclear element-1 (LINE-1), (6)-methylguanine-DNA methyltransferase (MGMT), and DNA mismatch repair gene human mutator L homologue 1 (hMLH1). AML-5 cells were treated with benzoquinone (BQ) and hydroquinone (HQ), and the promoter methylation of MGMT and hMLH1 was detected using the bisulfite sequencing PCR method. The degree of LINE-1 methylation in benzene-exposed workers was significantly lower than that of the controls (p<0.001), and the degree of MGMT (p<0.001) and hMLH1 (p = 0.01) methylation was significantly higher than that of the controls. The in vitro study validated the aberrant hypermethylation of hMLH1 after treatment with BQ. Among the cohort workers who were followed from 2009 to 2013, the LINE1 methylation elevated in 2013 than 2009 (p = 0.004), and premotor methylation in hMLH1 reduced in 2013 than 2009 (p = 0.045) with the reduction of the benzene exposure. This study provides evidence that benzene exposure can induce LINE-1 hypomethylation and DNA repair gene hypermethylation.
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Affiliation(s)
- Jingchao Ren
- Henan International Collaborative Laboratory for Health Effects and Intervention of Air Pollution, Medicine, School of Public Health, Xinxiang Medical University, Xinxiang, China
| | - Jun-peng Cui
- Henan International Collaborative Laboratory for Health Effects and Intervention of Air Pollution, Medicine, School of Public Health, Xinxiang Medical University, Xinxiang, China
| | - Mengkai Luo
- Henan International Collaborative Laboratory for Health Effects and Intervention of Air Pollution, Medicine, School of Public Health, Xinxiang Medical University, Xinxiang, China
| | - Huan Liu
- Henan International Collaborative Laboratory for Health Effects and Intervention of Air Pollution, Medicine, School of Public Health, Xinxiang Medical University, Xinxiang, China
| | - Pengfei Hao
- Xinxiang Center for Disease Control and Prevention, Xinxiang, China
| | - Xiao Wang
- Henan International Collaborative Laboratory for Health Effects and Intervention of Air Pollution, Medicine, School of Public Health, Xinxiang Medical University, Xinxiang, China
- * E-mail: (GZ); (XW)
| | - Guang-hui Zhang
- Henan International Collaborative Laboratory for Health Effects and Intervention of Air Pollution, Medicine, School of Public Health, Xinxiang Medical University, Xinxiang, China
- * E-mail: (GZ); (XW)
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27
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Ying X, Chen Y, Zheng Z, Duan S. Gout in males: a possible role for COMT hypomethylation. Clin Rheumatol 2019; 38:2865-2871. [PMID: 31165340 DOI: 10.1007/s10067-019-04607-0] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2018] [Revised: 04/27/2019] [Accepted: 05/15/2019] [Indexed: 12/22/2022]
Abstract
OBJECTIVE 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. METHODS 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). RESULTS 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%. CONCLUSION 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.
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Affiliation(s)
- Xiuru Ying
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang, 315211, China
| | - Yanfei Chen
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang, 315211, China
| | - Zhonghua Zheng
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang, 315211, China
| | - Shiwei Duan
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang, 315211, China.
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Singh N, Kazim SN, Sultana R, Tiwari D, Borkotoky R, Kakati S, Nath Das N, Kumar Saikia A, Bose S. Oxidative stress and deregulations in base excision repair pathway as contributors to gallbladder anomalies and carcinoma - a study involving North-East Indian population. Free Radic Res 2019; 53:473-485. [PMID: 31117842 DOI: 10.1080/10715762.2019.1606423] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Abstract
Gallbladder cancer (GBC) is a fatal condition with dismal prognosis and aggressive local invasiveness; and with uncharacterised molecular pathology relating to non-specific therapeutic modalities. Given the importance of oxidative stress in chronic diseases and carcinogenesis, and the lacunae in literature regarding its role in gallbladder diseases, this study aimed to study the involvement of oxidative stress and deregulation in the base excision repair (BER) pathway in the pathogenesis of gallbladder diseases including GBC. This study involved patients from the North-East Indian population, where the numbers of reported cases are increasing rapidly and alarmingly. Oxidative stress, based on 8-OH-dG levels, was found to be significantly higher in gallbladder anomalies (cholelithiasis [CL] and cholecystitis [CS]) and GBC at the plasma and DNA level, and was associated with GBC severity. The expressions of key BER pathway genes were downregulated in gallbladder anomalies and GBC compared to controls, and in GBC compared to both non-neoplastic controls and gallbladder anomalies. Expression of XRCC1 and hOGG1 was significantly associated with both susceptibility and severity of GBC. The XRCC1 codon280 polymorphism was associated with disease susceptibility; and significantly higher oxidative stress was observed in hOGG1 genotypic variants. The genomes of GBC patients were found to be more hypermethylated compared to controls, with the promoters of XRCC1 and hOGG1 being hypermethylated and, therefore, being silenced. This study underlined the prognostic significance of the oxidative stress marker 8-OH-dG and BER pathway genes, especially hOGG1 and XRCC1, in gallbladder anomalies and GBC, as well as stated their potential for therapeutic targeting.
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Affiliation(s)
- Nidhi Singh
- a Department of Biotechnology , Gauhati University , Guwahati , India
| | - Syed Naqui Kazim
- b Centre for Interdisciplinary Research in Basic Sciences , Jamia Millia Islamia , New Delhi , India
| | - Rizwana Sultana
- c Bioengineering and Technology , Gauhati University , Guwahati , India
| | - Diptika Tiwari
- c Bioengineering and Technology , Gauhati University , Guwahati , India
| | - Raktim Borkotoky
- a Department of Biotechnology , Gauhati University , Guwahati , India
| | | | | | - Anjan Kumar Saikia
- e Central Railway Hospital , Guwahati , India.,f GNRC Hospital , Guwahati , India
| | - Sujoy Bose
- a Department of Biotechnology , Gauhati University , Guwahati , India
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O'Keefe CM, Giammanco D, Li S, Pisanic TR, Wang THJ. Multilayer microfluidic array for highly efficient sample loading and digital melt analysis of DNA methylation. LAB ON A CHIP 2019; 19:444-451. [PMID: 30623957 PMCID: PMC6363116 DOI: 10.1039/c8lc01189c] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/07/2023]
Abstract
Liquid biopsies contain a treasure of genetic and epigenetic biomarkers that contain information for the detection and monitoring of human disease. DNA methylation is an epigenetic modification that is critical to determining cellular phenotype and often becomes altered in many disease states. In cancer, aberrant DNA methylation contributes to carcinogenesis and can profoundly affect tumor evolution, metastatic potential, and resistance to therapeutic intervention. However, current technologies are not well-suited for quantitative assessment of DNA methylation heterogeneity, especially in challenging samples such as liquid biopsies with low DNA input and high background. We present a multilayer microfluidic device for quantitative analysis of DNA methylation by digital PCR and high resolution melt (HRM). The multilayer design facilitates high-density array digitization aimed at maximizing sample loading efficiency. The platform achieves highly parallelized digital PCR-HRM-based discrimination of rare heterogeneous DNA methylation as low as 0.0001% methylated/unmethylated molecules of a classic tumor suppressor gene, CDKN2A (p14ARF).
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Affiliation(s)
- Christine M O'Keefe
- Department of Biomedical Engineering, Johns Hopkins School of Medicine, Baltimore, MD 21218, USA
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Mauger F, Deleuze JF. Technological advances in studying epigenetics biomarkers of prognostic potential for clinical research. PROGNOSTIC EPIGENETICS 2019:45-83. [DOI: 10.1016/b978-0-12-814259-2.00003-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
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Significant association between KDM1A promoter hypomethylation and colorectal cancer in Han Chinese. Pathol Res Pract 2018; 215:532-538. [PMID: 30638951 DOI: 10.1016/j.prp.2018.12.005] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/27/2018] [Revised: 11/15/2018] [Accepted: 12/05/2018] [Indexed: 02/06/2023]
Abstract
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.
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Menschikowski M, Jandeck C, Friedemann M, Nacke B, Hantsche S, Tiebel O, Sukocheva O, Hagelgans A. Identification of rare levels of methylated tumor DNA fragments using an optimized bias based pre-amplification-digital droplet PCR (OBBPA-ddPCR). Oncotarget 2018; 9:36137-36150. [PMID: 30546833 PMCID: PMC6281424 DOI: 10.18632/oncotarget.26315] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2018] [Accepted: 10/24/2018] [Indexed: 02/05/2023] Open
Abstract
BACKGROUND The analysis of aberrant DNA methylations is used for the diagnosis of cancer as significant changes in the gene methylation pattern are often detected during early carcinogenesis. In this study, we evaluated the performance of a two-step method that combines pre-amplification with ddPCR technique. RESULTS By using ddPCR, the dependence of amplification efficiency for methylated and unmethylated DNA fragments on the relevant MgCl2 concentration and the annealing temperature was established in addition to the primer design. We found that the efficiency can be adjusted toward methylated sequences by using primers covering one to four CpG sites under appropriately selected MgCl2 concentration and annealing temperature. Applying a PCR bias between 85% and 95%, five copies of methylated tumor DNA fragments were detected against a background of 700,000 copies of unmethylated DNA fragments with a high signal-to-noise ratio. The analysis of serum samples from patients with prostate cancer showed a significantly improved performance of the new method in comparison with the MS-HRM technique, ddPCR alone, or ddPCR in combination with an unbiased pre-amplification using methylation-independent primers. CONCLUSIONS We define this method as an optimized bias-based pre-amplification-digital droplet PCR (OBBPA-ddPCR) technique. This novel method is recommended for the early detection of cancer-specific DNA methylation biomarkers in the form of a liquid biopsy.
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Affiliation(s)
- Mario Menschikowski
- Institute of Clinical Chemistry and Laboratory Medicine, Carl Gustav Carus University Hospital, Technical University of Dresden, Dresden, Germany
| | - Carsten Jandeck
- Institute of Clinical Chemistry and Laboratory Medicine, Carl Gustav Carus University Hospital, Technical University of Dresden, Dresden, Germany
| | - Markus Friedemann
- Institute of Clinical Chemistry and Laboratory Medicine, Carl Gustav Carus University Hospital, Technical University of Dresden, Dresden, Germany
| | - Brit Nacke
- Institute of Clinical Chemistry and Laboratory Medicine, Carl Gustav Carus University Hospital, Technical University of Dresden, Dresden, Germany
| | - Saskia Hantsche
- Institute of Clinical Chemistry and Laboratory Medicine, Carl Gustav Carus University Hospital, Technical University of Dresden, Dresden, Germany
| | - Oliver Tiebel
- Institute of Clinical Chemistry and Laboratory Medicine, Carl Gustav Carus University Hospital, Technical University of Dresden, Dresden, Germany
| | - Olga Sukocheva
- School of Health Sciences, Flinders University of South Australia, Adelaide, Australia
| | - Albert Hagelgans
- Institute of Clinical Chemistry and Laboratory Medicine, Carl Gustav Carus University Hospital, Technical University of Dresden, Dresden, Germany
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Pan R, Zhou C, Dai J, Ying X, Yu H, Zhong J, Zhang Y, Wu B, Mao Y, Wu D, Ying J, Zhang W, Duan S. Endothelial PAS domain protein 1 gene hypomethylation is associated with colorectal cancer in Han Chinese. Exp Ther Med 2018; 16:4983-4990. [PMID: 30542453 PMCID: PMC6257466 DOI: 10.3892/etm.2018.6856] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2018] [Accepted: 08/23/2018] [Indexed: 12/17/2022] Open
Abstract
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%).
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Affiliation(s)
- Ranran Pan
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Cong Zhou
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Jie Dai
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Xiuru Ying
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Hang Yu
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Jie Zhong
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Yihan Zhang
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Boyi Wu
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Yiyi Mao
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Dongping Wu
- Department of Medical Oncology, Shaoxing People's Hospital, Shaoxing, Zhejiang 312000, P.R. China
| | - Jieer Ying
- Department of Medical Oncology, Zhejiang Cancer Hospital, Hangzhou, Zhejiang 310022, P.R. China
| | - Wei Zhang
- Department of Preventive Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.,The Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Shiwei Duan
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
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Zhou C, Pan R, Hu H, Li B, Dai J, Ying X, Yu H, Zhong J, Mao Y, Zhang Y, Wu D, Duan S. TNFRSF10C methylation is a new epigenetic biomarker for colorectal cancer. PeerJ 2018; 6:e5336. [PMID: 30225159 PMCID: PMC6139245 DOI: 10.7717/peerj.5336] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2018] [Accepted: 07/08/2018] [Indexed: 12/16/2022] Open
Abstract
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.
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Affiliation(s)
- Cong Zhou
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, China
| | - Ranran Pan
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, China
| | - Haochang Hu
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, China
| | - Bin Li
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, China
| | - Jie Dai
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, China
| | - Xiuru Ying
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, China
| | - Hang Yu
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, China
| | - Jie Zhong
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, China
| | - Yiyi Mao
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, China
| | - Yihan Zhang
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, China
| | - Dongping Wu
- Department of Medical Oncology, Shaoxing People's Hospital, Shaoxing Hospital of Zhejiang University, Zhejiang, China
| | - Shiwei Duan
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, China
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Harrison A, Pentieva K, Ozaki M, McNulty H, Parle-McDermott A. Assessment of candidate folate sensitive-differentially methylated regions in a randomised controlled trial of continued folic acid supplementation during the second and third trimesters of pregnancy. Ann Hum Genet 2018; 83:23-33. [PMID: 30175844 DOI: 10.1111/ahg.12281] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2018] [Revised: 07/06/2018] [Accepted: 07/20/2018] [Indexed: 11/29/2022]
Abstract
SCOPE The aim of this study was to identify if specific regions of the human genome were sensitive to folate status by displaying changes in their DNA methylation patterns in response to continued folic acid supplementation during pregnancy. METHODS AND RESULTS Samples (n = 119) from a previous randomised controlled trial in pregnancy were used to compare the DNA methylation profiles of the same woman pre- versus post-folic acid intervention. Candidate genes were identified from the literature and a pilot genome wide screen of six women (three from each of the folic acid and placebo arms of the trial). We did not observe consistent DNA methylation changes in response to folic acid intervention at any of our candidate genes (RASA4, DHFR, DHFR2, RASSF1A, EIF2C3, ATPF1). We did identify a 40% decrease in DNA methylation at the RASA4 promoter correlating with a 3.5-fold increase in its mRNA abundance in an in vitro cell culture model. CONCLUSION Continued folic acid intervention over a 22-week period did not appear to significantly influence the DNA methylation status of six candidate genes in blood samples of women compared to placebo. However, DNA methylation may play a role in the gene expression control of the RASA4 gene.
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Affiliation(s)
- Alan Harrison
- School of Biotechnology, Dublin City University, Dublin, Ireland
| | - Kristina Pentieva
- Nutrition Innovation Centre for Food & Health, Biomedical Sciences Research Institute, Ulster University, Coleraine, United Kingdom
| | - Mari Ozaki
- School of Biotechnology, Dublin City University, Dublin, Ireland
| | - Helene McNulty
- Nutrition Innovation Centre for Food & Health, Biomedical Sciences Research Institute, Ulster University, Coleraine, United Kingdom
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Pisanic TR, Cope LM, Lin SF, Yen TT, Athamanolap P, Asaka R, Nakayama K, Fader AN, Wang TH, Shih IM, Wang TL. Methylomic Analysis of Ovarian Cancers Identifies Tumor-Specific Alterations Readily Detectable in Early Precursor Lesions. Clin Cancer Res 2018; 24:6536-6547. [PMID: 30108103 DOI: 10.1158/1078-0432.ccr-18-1199] [Citation(s) in RCA: 45] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2018] [Revised: 07/12/2018] [Accepted: 08/09/2018] [Indexed: 12/22/2022]
Abstract
PURPOSE High-grade serous ovarian carcinoma (HGSOC) typically remains undiagnosed until advanced stages when peritoneal dissemination has already occurred. Here, we sought to identify HGSOC-specific alterations in DNA methylation and assess their potential to provide sensitive and specific detection of HGSOC at its earliest stages. EXPERIMENTAL DESIGN MethylationEPIC genome-wide methylation analysis was performed on a discovery cohort comprising 23 HGSOC, 37 non-HGSOC malignant, and 36 histologically unremarkable gynecologic tissue samples. The resulting data were processed using selective bioinformatic criteria to identify regions of high-confidence HGSOC-specific differential methylation. Quantitative methylation-specific real-time PCR (qMSP) assays were then developed for 8 of the top-performing regions and analytically validated in a cohort of 90 tissue samples. Lastly, qMSP assays were used to assess and compare methylation in 30 laser-capture microdissected (LCM) fallopian tube epithelia samples obtained from cancer-free and serous tubal intraepithelial carcinoma (STIC) positive women. RESULTS Bioinformatic selection identified 91 regions of robust, HGSOC-specific hypermethylation, 23 of which exhibited an area under the receiver-operator curve (AUC) value ≥ 0.9 in the discovery cohort. Seven of 8 top-performing regions demonstrated AUC values between 0.838 and 0.968 when analytically validated by qMSP in a 90-patient cohort. A panel of the 3 top-performing genes (c17orf64, IRX2, and TUBB6) was able to perfectly discriminate HGSOC (AUC 1.0). Hypermethylation within these loci was found exclusively in LCM fallopian tube epithelia from women with STIC lesions, but not in cancer-free fallopian tubes. CONCLUSIONS A panel of methylation biomarkers can be used to accurately identify HGSOC, even at precursor stages of the disease.
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Affiliation(s)
- Thomas R Pisanic
- Johns Hopkins Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, Maryland.
| | - Leslie M Cope
- Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.,Departments of Oncology and Biostatistics, Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Shiou-Fu Lin
- Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.,Departments of Gynecology and Obstetrics and Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Ting-Tai Yen
- Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.,Departments of Gynecology and Obstetrics and Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Pornpat Athamanolap
- Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Ryoichi Asaka
- Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.,Departments of Gynecology and Obstetrics and Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Kentaro Nakayama
- Department of Obstetrics and Gynecology, Shimane University School of Medicine, Izumo, Japan
| | - Amanda N Fader
- Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.,Departments of Gynecology and Obstetrics and Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Tza-Huei Wang
- Johns Hopkins Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, Maryland.,Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.,Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland.,Department of Mechanical Engineering, Johns Hopkins University, Baltimore, Maryland
| | - Ie-Ming Shih
- Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.,Departments of Gynecology and Obstetrics and Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Tian-Li Wang
- Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland. .,Departments of Gynecology and Obstetrics and Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland
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Liu L, Toung JM, Jassowicz AF, Vijayaraghavan R, Kang H, Zhang R, Kruglyak KM, Huang HJ, Hinoue T, Shen H, Salathia NS, Hong DS, Naing A, Subbiah V, Piha-Paul SA, Bibikova M, Granger G, Barnes B, Shen R, Gutekunst K, Fu S, Tsimberidou AM, Lu C, Eng C, Moulder SL, Kopetz ES, Amaria RN, Meric-Bernstam F, Laird PW, Fan JB, Janku F. Targeted methylation sequencing of plasma cell-free DNA for cancer detection and classification. Ann Oncol 2018; 29:1445-1453. [PMID: 29635542 PMCID: PMC6005020 DOI: 10.1093/annonc/mdy119] [Citation(s) in RCA: 87] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Background Targeted methylation sequencing of plasma cell-free DNA (cfDNA) has a potential to expand liquid biopsies to patients with tumors without detectable oncogenic alterations, which can be potentially useful in early diagnosis. Patients and methods We developed a comprehensive methylation sequencing assay targeting 9223 CpG sites consistently hypermethylated according to The Cancer Genome Atlas. Next, we carried out a clinical validation of our method using plasma cfDNA samples from 78 patients with advanced colorectal cancer, non-small-cell lung cancer (NSCLC), breast cancer or melanoma and compared results with patients' outcomes. Results Median methylation scores in plasma cfDNA samples from patients on therapy were lower than from patients off therapy (4.74 versus 85.29; P = 0.001). Of 68 plasma samples from patients off therapy, methylation scores detected the presence of cancer in 57 (83.8%), and methylation-based signatures accurately classified the underlying cancer type in 45 (78.9%) of these. Methylation scores were most accurate in detecting colorectal cancer (96.3%), followed by breast cancer (91.7%), melanoma (81.8%) and NSCLC (61.1%), and most accurate in classifying the underlying cancer type in colorectal cancer (88.5%), followed by NSCLC (81.8%), breast cancer (72.7%) and melanoma (55.6%). Low methylation scores versus high were associated with longer survival (10.4 versus 4.4 months, P < 0.001) and longer time-to-treatment failure (2.8 versus 1.6 months, P = 0.016). Conclusions Comprehensive targeted methylation sequencing of 9223 CpG sites in plasma cfDNA from patients with common advanced cancers detects the presence of cancer and underlying cancer type with high accuracy. Methylation scores in plasma cfDNA correspond with treatment outcomes.
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Affiliation(s)
- L Liu
- Illumina, Inc., San Diego, The University of Texas MD Anderson Cancer Center, Houston, USA.
| | - J M Toung
- Illumina, Inc., San Diego, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - A F Jassowicz
- Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, USA
| | - R Vijayaraghavan
- Illumina, Inc., San Diego, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - H Kang
- Illumina, Inc., San Diego, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - R Zhang
- Illumina, Inc., San Diego, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - K M Kruglyak
- Illumina, Inc., San Diego, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - H J Huang
- Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, USA
| | - T Hinoue
- Van Andel Research Institute, Grand Rapids, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - H Shen
- Van Andel Research Institute, Grand Rapids, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - N S Salathia
- Illumina, Inc., San Diego, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - D S Hong
- Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, USA
| | - A Naing
- Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, USA
| | - V Subbiah
- Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, USA
| | - S A Piha-Paul
- Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, USA
| | - M Bibikova
- Illumina, Inc., San Diego, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - G Granger
- Illumina, Inc., San Diego, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - B Barnes
- Illumina, Inc., San Diego, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - R Shen
- Illumina, Inc., San Diego, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - K Gutekunst
- Illumina, Inc., San Diego, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - S Fu
- Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, USA
| | - A M Tsimberidou
- Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, USA
| | - C Lu
- Department of Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - C Eng
- Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - S L Moulder
- Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - E S Kopetz
- Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - R N Amaria
- Department of Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - F Meric-Bernstam
- Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, USA
| | - P W Laird
- Van Andel Research Institute, Grand Rapids, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - J-B Fan
- Illumina, Inc., San Diego, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - F Janku
- Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, USA.
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Hypermethylation of MDFI promoter with NSCLC is specific for females, non-smokers and people younger than 65. Oncol Lett 2018; 15:9017-9024. [PMID: 29805634 PMCID: PMC5958687 DOI: 10.3892/ol.2018.8535] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2017] [Accepted: 01/25/2018] [Indexed: 01/03/2023] Open
Abstract
Non-small cell lung carcinoma (NSCLC) is a major subtype of lung cancer. Aberrant DNA methylation has been frequently observed in NSCLC. The aim of the present study was to investigate the role of MyoD family inhibitor (MDFI) methylation in NSCLC. Formalin-fixed paraffin-embedded tumor tissues and adjacent non-cancerous tissues were collected from a total of 111 patients with NSCLC. A methylation assay was performed using the quantitative methylation-specific polymerase chain reaction method. The percentage of methylated reference was used to represent the methylation level of the MDFI promoter. Data mining of a dataset from The Cancer Genome Atlas (TCGA) demonstrated that MDFI promoter methylation levels were significantly increased in 830 tumor tissues compared with 75 non-tumor tissues (P=0.012). However, the results on tissues obtained in the present study indicated that the MDFI promoter methylation levels in tumor tissues were not significantly different compared with those in the adjacent non-tumor tissues (P=0.159). Subsequent breakdown analysis identified that higher MDFI promoter methylation levels were significantly associated with NSCLC in females (P=0.031), but not in males (P=0.832). Age-based subgroup analysis demonstrated that higher MDFI promoter methylation levels were significantly associated with NSCLC in younger patients (≤65 years; P=0.003), but not in older patients (P=0.327). In addition, the association of MDFI methylation with NSCLC was significant in non-smokers (P=0.014), but not in smokers (P=0.832). Similar results also have been determined from subgroup analysis of the TCGA datasets. The Gene Expression Omnibus database indicated MDFI expression restoration in partial lung cancer cell lines (H1299 and Hotz) following demethylation treatment. However, it was identified that MDFI promoter hypermethylation was not significantly associated with prognosis of NSCLC (P>0.05). In conclusion, the present study indicated that the association of higher methylation of the MDFI promoter with NSCLC may be specific to females, non-smokers and people aged ≤65.
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Misawa K, Imai A, Mochizuki D, Mima M, Endo S, Misawa Y, Kanazawa T, Mineta H. Association of TET3 epigenetic inactivation with head and neck cancer. Oncotarget 2018; 9:24480-24493. [PMID: 29849955 PMCID: PMC5966249 DOI: 10.18632/oncotarget.25333] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2017] [Accepted: 04/21/2018] [Indexed: 12/22/2022] Open
Abstract
The aim of this study was to clarify the epigenetic regulation of ten eleven translocation protein (TET) family genes, which can provide insights into the mechanisms of tumorigenesis and the risk of disease recurrence in head and neck squamous cell carcinoma (HNSCC). We generated methylation profiles of TET1, TET2 and TET3 genes in tumor samples obtained from 233 patients with HNSCC; these included 57 hypopharynx, 44 larynx, 69 oral cavity, and 63 oropharynx tumor samples. The mRNA expression and promoter DNA methylation of TET family genes were examined via quantitative RT-PCR and methylation-specific PCR, respectively. Promoter methylation was compared with various clinical characteristics and the TET methylation index (TE-MI). The TE-MI, representing the number of methylation events in TET family genes, was positively correlated with alcohol consumption (P = 0.004), high-risk human papilloma virus (HPV) status (P = 0.004) and disease recurrence (P = 0.002). The simultaneous methylation analysis of TET family genes was correlated with reduced disease-free survival in unfavorable event groups (log-rank test, P = 0.026). In the multivariate Cox proportional hazards analysis, TET3 methylation in T1 and T2 tumor stages, oropharyngeal cancer, and oral cancer patients exhibited high association with poor survival (hazard ratio: 2.64, P = 0.014; 3.55, P = 0.048; 2.63, P = 0.028, respectively). A joint analysis of the tumor suppressor gene methylation index showed a significant trend toward a higher TE-MI. The methylation status of TET3 was independently associated with aggressive tumor behavior and a global effect on DNA methylation status in HNSCC.
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Affiliation(s)
- Kiyoshi Misawa
- Department of Otolaryngology, Head and Neck Surgery, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Atsushi Imai
- Department of Otolaryngology, Head and Neck Surgery, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Daiki Mochizuki
- Department of Otolaryngology, Head and Neck Surgery, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Masato Mima
- Department of Otolaryngology, Head and Neck Surgery, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Shiori Endo
- Department of Otolaryngology, Head and Neck Surgery, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Yuki Misawa
- Department of Otolaryngology, Head and Neck Surgery, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Takeharu Kanazawa
- Department of Otolaryngology, Head and Neck Surgery, Jichi Medical University, Tochigi, Japan
| | - Hiroyuki Mineta
- Department of Otolaryngology, Head and Neck Surgery, Hamamatsu University School of Medicine, Shizuoka, Japan
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Mauger F, Kernaleguen M, Lallemand C, Kristensen VN, Deleuze JF, Tost J. Enrichment of methylated molecules using enhanced-ice-co-amplification at lower denaturation temperature-PCR (E-ice-COLD-PCR) for the sensitive detection of disease-related hypermethylation. Epigenomics 2018; 10:525-537. [PMID: 29697281 DOI: 10.2217/epi-2017-0166] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
AIM The detection of specific DNA methylation patterns bears great promise as biomarker for personalized management of cancer patients. Co-amplification at lower denaturation temperature-PCR (COLD-PCR) assays are sensitive methods, but have previously only been able to analyze loss of DNA methylation. MATERIALS & METHODS Enhanced (E)-ice-COLD-PCR reactions starting from 2 ng of bisulfite-converted DNA were developed to analyze methylation patterns in two promoters with locked nucleic acid (LNA) probes blocking amplification of unmethylated CpGs. The enrichment of methylated molecules was compared to quantitative (q)PCR and quantified using serial dilutions. RESULTS E-ice-COLD-PCR allowed the multiplexed enrichment and quantification of methylated DNA. Assays were validated in primary breast cancer specimens and circulating cell-free DNA from cancer patients. CONCLUSION E-ice-COLD-PCR could prove a useful tool in the context of DNA methylation analysis for personalized medicine.
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Affiliation(s)
- Florence Mauger
- Laboratory for Epigenetics & Environment, Centre National de Recherche en Génomique Humaine, CEA-Institut de Biologie François Jacob, Evry, France
| | - Magali Kernaleguen
- Laboratory for Epigenetics & Environment, Centre National de Recherche en Génomique Humaine, CEA-Institut de Biologie François Jacob, Evry, France
| | - Céline Lallemand
- Laboratory for Epigenetics & Environment, Centre National de Recherche en Génomique Humaine, CEA-Institut de Biologie François Jacob, Evry, France
| | - Vessela N Kristensen
- Department of Cancer Genetics, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Oslo, Norway.,Department of Clinical Molecular Biology & Laboratory Science (EpiGen), Akershus University Hospital, Division of Medicine, 1476 Lørenskog, Norway.,Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway
| | - Jean-François Deleuze
- Laboratory for Epigenetics & Environment, Centre National de Recherche en Génomique Humaine, CEA-Institut de Biologie François Jacob, Evry, France.,Centre d'Etudes du Polymorphisme Humain, CEPH-Fondation Jean Dausset, Paris, France.,Laboratoire d'Excellence GenMed, France
| | - Jörg Tost
- Laboratory for Epigenetics & Environment, Centre National de Recherche en Génomique Humaine, CEA-Institut de Biologie François Jacob, Evry, France
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Hu Z, Yang Y, Zhao Y, Yu H, Ying X, Zhou D, Zhong J, Zheng Z, Liu J, Pan R, Zhang W, Cheng F, Duan S. APOE hypermethylation is associated with autism spectrum disorder in a Chinese population. Exp Ther Med 2018; 15:4749-4754. [PMID: 29844799 PMCID: PMC5958870 DOI: 10.3892/etm.2018.6069] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2017] [Accepted: 03/01/2018] [Indexed: 12/27/2022] Open
Abstract
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.
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Affiliation(s)
- Zhenyu Hu
- Department of Child Psychiatry, Ningbo Kangning Hospital, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Yong Yang
- Zhejiang Provincial Key Laboratory of Pathophysiology, Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Yuanzhi Zhao
- Department of Child Psychiatry, Ningbo Kangning Hospital, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Hang Yu
- Zhejiang Provincial Key Laboratory of Pathophysiology, Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Xiuru Ying
- Zhejiang Provincial Key Laboratory of Pathophysiology, Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Dongsheng Zhou
- Department of Child Psychiatry, Ningbo Kangning Hospital, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Jie Zhong
- Zhejiang Provincial Key Laboratory of Pathophysiology, Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Zhonghua Zheng
- Zhejiang Provincial Key Laboratory of Pathophysiology, Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Jing Liu
- Zhejiang Provincial Key Laboratory of Pathophysiology, Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Ranran Pan
- Zhejiang Provincial Key Laboratory of Pathophysiology, Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Wenwu Zhang
- Department of Child Psychiatry, Ningbo Kangning Hospital, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Fang Cheng
- Department of Child Psychiatry, Ningbo Kangning Hospital, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
| | - Shiwei Duan
- Zhejiang Provincial Key Laboratory of Pathophysiology, Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China
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Misawa K, Misawa Y, Imai A, Mochizuki D, Endo S, Mima M, Ishikawa R, Kawasaki H, Yamatodani T, Kanazawa T. Epigenetic modification of SALL1 as a novel biomarker for the prognosis of early stage head and neck cancer. J Cancer 2018; 9:941-949. [PMID: 29581773 PMCID: PMC5868161 DOI: 10.7150/jca.23527] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2017] [Accepted: 01/31/2018] [Indexed: 12/12/2022] Open
Abstract
This study examined Sal-like protein (SALL)1 methylation profiles in head and neck squamous-cell carcinoma (HNSCC) patients at diagnosis and follow-up, and evaluated their prognostic significance and value as a biomarker. SALL1 expression was examined in a panel of cell lines by quantitative reverse transcription PCR (qRT-PCR). Promoter methylation was determined by quantitative methylation-specific polymerase chain reaction (qMSP) and was compared to the clinical characteristics of 205 samples. SALL1 promoter methylation was associated with transcriptional inhibition and was correlated with disease recurrence in 31.7% of cases, with an odds ratio of 1.694 (95% confidence interval: 1.093-2.626; P = 0.018) by multivariate Cox proportional hazard regression analysis. SALL1 promoter hypermethylation showed highly discriminatory receiver operator characteristic curve profiles that clearly distinguished HNSCC from adjacent normal mucosal tissue, and was correlated with reduced disease-free survival in early stage T1 and T2 patients (log-rank test, P < 0.001). SALL1 methylation was significantly correlated with the methylation status of both SALL3 and CDH1. This study suggests that CpG hypermethylation is a likely mechanism of SALL1 gene inactivation, supporting the hypothesis that SALL1 might play a role in HNSCC tumorigenesis and could serve as an important biomarker.
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Affiliation(s)
- Kiyoshi Misawa
- Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan
| | - Yuki Misawa
- Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan
| | - Atsushi Imai
- Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan
| | - Daiki Mochizuki
- Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan
| | - Shiori Endo
- Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan
| | - Masato Mima
- Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan
| | - Ryuji Ishikawa
- Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan
| | - Hideya Kawasaki
- Department of Regenerative & Infectious Pathology, Hamamatsu University School of Medicine, Hamamatsu, Japan
| | - Takashi Yamatodani
- Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan
| | - Takeharu Kanazawa
- Department of Otolaryngology/Head and Neck Surgery, Jichi Medical University, Tochigi, Japan
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Misawa K, Mochizuki D, Imai A, Endo S, Mima M, Misawa Y, Kanazawa T, Carey TE, Mineta H. Prognostic value of aberrant promoter hypermethylation of tumor-related genes in early-stage head and neck cancer. Oncotarget 2018; 7:26087-98. [PMID: 27027429 PMCID: PMC5041966 DOI: 10.18632/oncotarget.8317] [Citation(s) in RCA: 42] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2015] [Accepted: 03/11/2016] [Indexed: 01/26/2023] Open
Abstract
Staging and pathological grading are useful, but imperfect predictors of recurrence in head and neck squamous cell carcinoma (HNSCC). Accordingly, molecular biomarkers that predict the risk of recurrence are necessary to improve clinical outcomes. The methylation statuses of the promoters of 11 tumor-related genes (p16, RASSF1A, E-cadherin, H-cadherin, MGMT, DAPK, DCC, COL1A2, TAC1, SST, and GALR1) were analyzed in 133 HNSCC cases using quantitative methylation-specific PCR. We detected frequent methylation of p16 (44%), RASSF1A (18%), E-cadherin (53%), H-cadherin (35%), MGMT (35%), DAPK (53%), DCC (42%), COL1A2 (44%), TAC1 (61%), SST (64%), and GALR1 (44%) in HNSCC. Disease-free survival was lower in patients with 6–11 methylated genes than in those with 0–5 methylated genes (log-rank test, P = 0.001). In a multivariate Cox proportional hazards analysis, the methylation of E-cadherin, COL1A2, TAC1, and GALR1 was associated with poor survival, with hazard ratios of 4.474 (95% CI, 1.241–16.124). In a joint analysis of these four genes, patients with 2–4 methylated genes had a significantly lower survival rate than those with 0–1 methylated genes in early-stage HNSCC. Importantly, the methylation of some genes was closely related to poor prognosis in early-stage HNSCC, providing strong evidence that these hypermethylated genes are valuable biomarkers for prognostic evaluation.
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Affiliation(s)
- Kiyoshi Misawa
- Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Daiki Mochizuki
- Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Atsushi Imai
- Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Shiori Endo
- Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Masato Mima
- Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Yuki Misawa
- Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Takeharu Kanazawa
- Department of Otolaryngology/Head and Neck Surgery, Jichi Medical University, Tochigi, Japan
| | - Thomas E Carey
- Department of Otolaryngology/Head and Neck Surgery, Laboratory of Head and Neck Cancer Biology, University of Michigan, Ann Arbor, MI, USA
| | - Hiroyuki Mineta
- Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Shizuoka, Japan
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Huang M, Zhou X, Wang H, Xing D. Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Triggered Isothermal Amplification for Site-Specific Nucleic Acid Detection. Anal Chem 2018; 90:2193-2200. [PMID: 29260561 DOI: 10.1021/acs.analchem.7b04542] [Citation(s) in RCA: 189] [Impact Index Per Article: 27.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
A novel CRISPR/Cas9 triggered isothermal exponential amplification reaction (CAS-EXPAR) strategy based on CRISPR/Cas9 cleavage and nicking endonuclease (NEase) mediated nucleic acids amplification was developed for rapid and site-specific nucleic acid detection. CAS-EXPAR was primed by the target DNA fragment produced by cleavage of CRISPR/Cas9, and the amplification reaction performed cyclically to generate a large number of DNA replicates which were detected using a real-time fluorescence monitoring method. This strategy that combines the advantages of CRISPR/Cas9 and exponential amplification showed high specificity as well as rapid amplification kinetics. Unlike conventional nucleic acids amplification reactions, CAS-EXPAR does not require exogenous primers, which often cause target-independent amplification. Instead, primers are first generated by Cas9/sgRNA directed site-specific cleavage of target and accumulated during the reaction. It was demonstrated this strategy gave a detection limit of 0.82 amol and showed excellent specificity in discriminating single-base mismatch. Moreover, the applicability of this method to detect DNA methylation and L. monocytogenes total RNA was also verified. Therefore, CAS-EXPAR may provide a new paradigm for efficient nucleic acid amplification and hold the potential for molecular diagnostic applications.
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Affiliation(s)
- Mengqi Huang
- MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University , Guangzhou 510631, PR China
| | - Xiaoming Zhou
- MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University , Guangzhou 510631, PR China
| | - Huiying Wang
- MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University , Guangzhou 510631, PR China
| | - Da Xing
- MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University , Guangzhou 510631, PR China
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Crime investigation through DNA methylation analysis: methods and applications in forensics. EGYPTIAN JOURNAL OF FORENSIC SCIENCES 2018. [DOI: 10.1186/s41935-018-0042-1] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
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Misawa K, Mochizuki D, Imai A, Mima M, Misawa Y, Mineta H. Analysis of Site-Specific Methylation of Tumor-Related Genes in Head and Neck Cancer: Potential Utility as Biomarkers for Prognosis. Cancers (Basel) 2018; 10:cancers10010027. [PMID: 29361757 PMCID: PMC5789377 DOI: 10.3390/cancers10010027] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2017] [Revised: 01/18/2018] [Accepted: 01/19/2018] [Indexed: 12/14/2022] Open
Abstract
Clarifying the epigenetic regulation of tumor-related genes (TRGs) can provide insights into the mechanisms of tumorigenesis and the risk for disease recurrence in HPV-negative head and neck cancers, originating in the hypopharynx, larynx, and oral cavity. We analyzed the methylation status of the promoters of 30 TRGs in 178 HPV-negative head and neck cancer patients using a quantitative methylation-specific PCR. Promoter methylation was correlated with various clinical characteristics and patient survival. The mean number of methylated TRGs was 14.2 (range, 2-25). In the multivariate Cox proportional hazards analysis, the methylation of COL1A2 and VEGFR1 was associated with poor survival for hypopharyngeal cancer, with hazard ratios: 3.19; p = 0.009 and 3.07; p = 0.014, respectively. The methylation of p16 and COL1A2 were independent prognostic factors for poor survival in laryngeal cancer (hazard ratio: 4.55; p = 0.013 and 3.12; p = 0.035, respectively). In patients with oral cancer, the methylation of TAC1 and SSTR1 best correlated with poor survival (hazard ratio: 4.29; p = 0.005 and 5.38; p = 0.029, respectively). Our findings suggest that methylation status of TRGs could serve as important site-specific biomarkers for prediction of clinical outcomes in patients with HPV-negative head and neck cancer.
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Affiliation(s)
- Kiyoshi Misawa
- Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Shizuoka 431-3192, Japan.
| | - Daiki Mochizuki
- Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Shizuoka 431-3192, Japan.
| | - Atsushi Imai
- Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Shizuoka 431-3192, Japan.
| | - Masato Mima
- Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Shizuoka 431-3192, Japan.
| | - Yuki Misawa
- Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Shizuoka 431-3192, Japan.
| | - Hiroyuki Mineta
- Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Shizuoka 431-3192, Japan.
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Sun Y, Sun Y, Tian W, Liu C, Gao K, Li Z. A novel restriction endonuclease GlaI for rapid and highly sensitive detection of DNA methylation coupled with isothermal exponential amplification reaction. Chem Sci 2017; 9:1344-1351. [PMID: 29675182 PMCID: PMC5887237 DOI: 10.1039/c7sc04975g] [Citation(s) in RCA: 67] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2017] [Accepted: 12/09/2017] [Indexed: 12/22/2022] Open
Abstract
Sensitive and accurate detection of site-specific DNA methylation is of critical significance for early diagnosis of human diseases, especially cancers. Herein, for the first time we employ a novel methylation-dependent restriction endonuclease GlaI to detect site-specific DNA methylation in a highly specific and sensitive way by coupling with isothermal exponential amplification reaction (EXPAR). GlaI can only cut the methylated target site with excellent selectivity but leave the unmethylated DNA intact. Then the newly exposed end fragments of methylated DNA can trigger EXPAR for highly efficient signal amplification while the intact unmethylated DNA will not initiate EXPAR at all. As such, only the methylated DNA is quantitatively and faithfully reflected by the real-time fluorescence signal of the GlaI-EXPAR system, and the potential false positive interference from unmethylated DNA can be effectively eliminated. Therefore, by integrating the unique features of GlaI for highly specific methylation discrimination and EXPAR for rapid and powerful signal amplification, the elegant GlaI-EXPAR assay allows the direct quantification of methylated DNA with ultrahigh sensitivity and accuracy. The detection limit of methylated DNA target has been pushed down to the aM level and the whole detection process of GlaI-EXPAR can be accomplished within a short time of 2 h. More importantly, ultrahigh specificity is achieved and as low as 0.01% methylated DNA can be clearly identified in the presence of a large excess of unmethylated DNA. This GlaI-EXPAR is also demonstrated to be capable of determining site-specific DNA methylations in real genomic DNA samples. Sharing the distinct advantages of ultrahigh sensitivity, outstanding specificity and facile operation, this new GlaI-EXPAR strategy may provide a robust and reliable platform for the detection of site-specific DNA methylations with low abundances.
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Affiliation(s)
- Yueying Sun
- Key Laboratory of Applied Surface and Colloid Chemistry , Ministry of Education , Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province , School of Chemistry and Chemical Engineering , Shaanxi Normal University , Xi'an 710062 , Shaanxi Province , P. R. China . ;
| | - Yuanyuan Sun
- Key Laboratory of Applied Surface and Colloid Chemistry , Ministry of Education , Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province , School of Chemistry and Chemical Engineering , Shaanxi Normal University , Xi'an 710062 , Shaanxi Province , P. R. China . ;
| | - Weimin Tian
- Key Laboratory of Applied Surface and Colloid Chemistry , Ministry of Education , Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province , School of Chemistry and Chemical Engineering , Shaanxi Normal University , Xi'an 710062 , Shaanxi Province , P. R. China . ;
| | - Chenghui Liu
- Key Laboratory of Applied Surface and Colloid Chemistry , Ministry of Education , Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province , School of Chemistry and Chemical Engineering , Shaanxi Normal University , Xi'an 710062 , Shaanxi Province , P. R. China . ;
| | - Kejian Gao
- Key Laboratory of Applied Surface and Colloid Chemistry , Ministry of Education , Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province , School of Chemistry and Chemical Engineering , Shaanxi Normal University , Xi'an 710062 , Shaanxi Province , P. R. China . ;
| | - Zhengping Li
- Key Laboratory of Applied Surface and Colloid Chemistry , Ministry of Education , Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province , School of Chemistry and Chemical Engineering , Shaanxi Normal University , Xi'an 710062 , Shaanxi Province , P. R. China . ;
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Oh TJ, Oh HI, Seo YY, Jeong D, Kim C, Kang HW, Han YD, Chung HC, Kim NK, An S. Feasibility of quantifying SDC2 methylation in stool DNA for early detection of colorectal cancer. Clin Epigenetics 2017; 9:126. [PMID: 29225717 PMCID: PMC5715626 DOI: 10.1186/s13148-017-0426-3] [Citation(s) in RCA: 70] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2017] [Accepted: 11/23/2017] [Indexed: 01/04/2023] Open
Abstract
Background 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 SDC2 were aberrantly methylated in tumor tissues of most CRC patients through comprehensive methylation analysis and demonstrated a high potential of quantification of SDC2 methylation in blood for early detection of colorectal cancer. In this study, we aim to investigate the feasibility of quantifying SDC2 methylation in stool DNA for the early detection of CRC. The objective of this study was to confirm a high frequency of SDC2 methylation in tumor tissues at various stages of CRC and investigate the feasibility of a quantitative test for SDC2 methylation in fecal DNA by highly sensitive and accurate real-time PCR for early detection of CRC. Methods Bisulfite-pyrosequencing assay was performed to measure the SDC2 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 SDC2 and quantitative methylation-specific real time PCR (qMSP) for SDC2, named as meSDC2 LTE-qMSP assay. Its limit of detection was 0.1% methylation (corresponding to ~ 6 copies in total ~ 6200 genome copies). Results Positive SDC2 methylation was observed in 100% of primary tumors, 90.6% of adenomatous polyps, 94.1% of hyperplastic polyps, and 0% of normal tissues. SDC2 methylation level also significantly (P < 0.01) increased according to the severity of lesions. In stool DNA test for SDC2 methylation by LTE-qMSP comparing CRC patients with various stages (I to IV) (n = 50) and precancerous lesions (n = 21) with healthy subjects (n = 22), the overall sensitivity was 90.0% for detecting CRC and 33.3% for detecting small polyps, with a specificity of 90.9%. Conclusions Taken together, our result indicates that stool DNA-based SDC2 methylation test by LTE-qMSP is a potential noninvasive diagnostic tool for early detection of CRC.
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Affiliation(s)
- Tae Jeong Oh
- Genomictree, Inc, 44-6 Techno 10-ro Yuseong-gu, Daejeon, 34027 South Korea
| | - Hyun Il Oh
- Genomictree, Inc, 44-6 Techno 10-ro Yuseong-gu, Daejeon, 34027 South Korea
| | - Yang Yei Seo
- Genomictree, Inc, 44-6 Techno 10-ro Yuseong-gu, Daejeon, 34027 South Korea
| | - Dongjun Jeong
- Department of Pathology, College of Medicine, Soonchunhyang University, 23-20 Byeongmyeong-dong Dongnam-gu, Cheonan, Chungcheongnam-do 31151 South Korea
| | - Changjin Kim
- Department of Pathology, College of Medicine, Soonchunhyang University, 23-20 Byeongmyeong-dong Dongnam-gu, Cheonan, Chungcheongnam-do 31151 South Korea
| | - Hyoun Woo Kang
- Department of Internal Medicine, Dongguk University Ilsan Hospital, College of Medicine, Dongguk University, 27 Dongguk-ro Ilsandong-gu, Goyang-si, Gyeonggi-do 10326 South Korea
| | - Yoon Dae Han
- Department of Surgery, Yonsei University College of Medicine, 50-1 Yonsei-ro Seodaemun-gu, Seoul, 03722 South Korea
| | - Hyun Cheol Chung
- Yonsei Cancer Center Yonsei University College of Medicine, 50-1 Yonsei-ro Seodaemun-gu, Seoul, 03722 South Korea
| | - Nam Kyu Kim
- Department of Surgery, Yonsei University College of Medicine, 50-1 Yonsei-ro Seodaemun-gu, Seoul, 03722 South Korea
| | - Sungwhan An
- Genomictree, Inc, 44-6 Techno 10-ro Yuseong-gu, Daejeon, 34027 South Korea
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Li B, Chen X, Jiang Y, Yang Y, Zhong J, Zhou C, Hu H, Duan S. CCL2 promoter hypomethylation is associated with gout risk in Chinese Han male population. Immunol Lett 2017; 190:15-19. [DOI: 10.1016/j.imlet.2017.06.011] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2017] [Revised: 06/25/2017] [Accepted: 06/30/2017] [Indexed: 12/27/2022]
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Hu H, Chen X, Wang C, Jiang Y, Li J, Ying X, Yang Y, Li B, Zhou C, Zhong J, Wu D, Ying J, Duan S. The role of TFPI2 hypermethylation in the detection of gastric and colorectal cancer. Oncotarget 2017; 8:84054-84065. [PMID: 29137404 PMCID: PMC5663576 DOI: 10.18632/oncotarget.21097] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2016] [Accepted: 08/28/2017] [Indexed: 02/06/2023] Open
Abstract
Gastrointestinal cancer is a prevalent disease with high morbidity and mortality. Tissue factor pathway inhibitor 2 (TFPI2) gene could protect the extracellular matrix of cancer cells from degradation and tumor invasion. The goal of our study was to estimate the diagnostic value of TFPI2 hypermethylation in gastric cancer (GC) and colorectal cancer (CRC). TFPI2 methylation was measured by quantitative methylation-specific polymerase chain reaction (qMSP) method in 114 GC and 80 CRC tissues and their paired non-tumor tissues. Our results showed that TFPI2 methylation was significantly higher in tumor tissues (GC: 29.940% vs. 12.785%, P < 0.001; CRC: 26.930% vs. 5.420%, P < 0.001). The methylation level of TFPI2 in colorectal tumor tissues was significantly higher than that in colorectal normal tissues (26.930% versus 0.002%, P < 0.00001). In GC, TFPI2 hypermethylation yielded an area under the curve (AUC) of 0.762 (95% CI: 0.696–0.828) with a sensitivity of 68% and a specificity of 83%. In CRC, TFPI2 hypermethylation yielded an AUC of 0.759 (95% CI: 0.685–0.834) with a sensitivity of 61% and a specificity of 84%. Similarly, TCGA data also supported TFPI2 hypermethylation was a promising diagnostic marker for GC and CRC. Moreover, the dual-luciferase reporter assay showed TFPI2 fragment could upregulate gene expression (fold change = 5, P = 0.005). Data mining further indicated that TFPI2 expression in CRC cell lines was significantly increased after 5’-AZA-deoxycytidine treatment (fold change > 1.37). In conclusion, TFPI2 hypermethylation might be a promising diagnostic biomarker for GC and CRC.
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Affiliation(s)
- Haochang Hu
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, China
| | - Xiaoying Chen
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, China
| | - Cheng Wang
- Department of Medical Oncology, Shaoxing People's Hospital, Shaoxing Hospital of Zhejiang University, Zhejiang 312000, China
| | - Yuting Jiang
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, China
| | - Jingjing Li
- Department of Medical Oncology, Zhejiang Cancer Hospital, Hangzhou, Zhejiang 310022, China
| | - Xiuru Ying
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, China
| | - Yong Yang
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, China
| | - Bin Li
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, China
| | - Cong Zhou
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, China
| | - Jie Zhong
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, China
| | - Dongping Wu
- Department of Medical Oncology, Shaoxing People's Hospital, Shaoxing Hospital of Zhejiang University, Zhejiang 312000, China
| | - Jieer Ying
- Department of Medical Oncology, Zhejiang Cancer Hospital, Hangzhou, Zhejiang 310022, China
| | - Shiwei Duan
- Medical Genetics Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, China
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