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1
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van Vliet MM, Boers RG, Boers JB, Schäffers OJM, van der Meeren LE, Steegers‐Theunissen RPM, Gribnau J, Schoenmakers S. Genome-wide methylation profiling of maternal cell-free DNA using methylated DNA sequencing (MeD-seq) indicates a placental and immune-cell signature. Eur J Clin Invest 2025; 55:e14363. [PMID: 39589853 PMCID: PMC11810559 DOI: 10.1111/eci.14363] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/03/2024] [Accepted: 11/13/2024] [Indexed: 11/28/2024]
Abstract
BACKGROUND Placental-originated cell-free DNA (cfDNA) provides unique opportunities to study (epi)genetic placental programming remotely, but studies investigating the cfDNA methylome are scarce and usually technologically challenging. Methylated DNA sequencing (MeD-seq) is well compatible with low cfDNA concentrations and has a high genome-wide coverage. We therefore aim to investigate the feasibility of genome-wide methylation profiling of first trimester maternal cfDNA using MeD-seq, by identifying placental-specific methylation marks in cfDNA. METHODS We collected cfDNA from nonpregnant controls (female n = 6, male n = 12) and pregnant women (n = 10), first trimester placentas (n = 10), and paired preconceptional and first trimester buffy coats (total n = 20). Differentially methylated regions (DMRs) were identified between pregnant and nonpregnant women. We investigated placental-specific markers in maternal cfDNA, including RASSF1 promoter and Y-chromosomal methylation, and studied overlap with placental and buffy coat DNA methylation. RESULTS We identified 436 DMRs between cfDNA from pregnant and nonpregnant women, which were validated using male cfDNA. RASSF1 promoter methylation was higher in maternal cfDNA (fold change 2.87, unpaired t-test p < .0001). Differential methylation of Y-chromosomal sequences could determine fetal sex. DMRs in maternal cfDNA showed large overlap with DNA methylation of these regions in placentas and buffy coats. Sixteen DMRs in maternal cfDNA were specifically found only in placentas. These novel potential placental-specific DMRs were more prominent than RASSF1. CONCLUSIONS MeD-seq can detect (novel) genome-wide placental DNA methylation marks and determine fetal sex in maternal cfDNA. Our results indicate a placental and immune-cell contribution to the pregnancy-specific cfDNA methylation signature. This study supports future research into maternal cfDNA methylation.
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Affiliation(s)
- Marjolein M. van Vliet
- Department of Obstetrics and GynaecologyErasmus MCRotterdamThe Netherlands
- Department of Developmental BiologyErasmus MCRotterdamThe Netherlands
| | - Ruben G. Boers
- Department of Developmental BiologyErasmus MCRotterdamThe Netherlands
| | - Joachim B. Boers
- Department of Developmental BiologyErasmus MCRotterdamThe Netherlands
| | - Olivier J. M. Schäffers
- Department of Obstetrics and GynaecologyErasmus MCRotterdamThe Netherlands
- Department of Developmental BiologyErasmus MCRotterdamThe Netherlands
| | - Lotte E. van der Meeren
- Department of PathologyErasmus Medical Centre RotterdamRotterdamThe Netherlands
- Department of PathologyLeiden University Medical CenterLeidenThe Netherlands
| | | | - Joost Gribnau
- Department of Developmental BiologyErasmus MCRotterdamThe Netherlands
| | - Sam Schoenmakers
- Department of Obstetrics and GynaecologyErasmus MCRotterdamThe Netherlands
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2
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Zhao M, Cai M, Lei F, Yuan X, Liu Q, Fang Y, Zhu B. AI-driven feature selection and epigenetic pattern analysis: A screening strategy of CpGs validated by pyrosequencing for body fluid identification. Forensic Sci Int 2024; 367:112339. [PMID: 39729807 DOI: 10.1016/j.forsciint.2024.112339] [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: 08/29/2024] [Revised: 12/01/2024] [Accepted: 12/06/2024] [Indexed: 12/29/2024]
Abstract
Identification of body fluid stain at crime scene is one of the important tasks of forensic evidence analysis. Currently, body fluid-specific CpGs detected by DNA methylation microarray screening, have been widely studied for forensic body fluid identification. However, some CpGs have limited ability to distinguish certain body fluid types. The ongoing need is to discover novel methylation markers and fully validate them to enhance their evidentiary strength in complex forensic scenarios. This research gathered forensic-related DNA methylation microarrays data from the Gene Expression Omnibus (GEO) database. A novel screening strategy for marker selection was developed, combining feature selection algorithms (elastic net, information gain ratio, feature importance based on Random Forest, and mutual information coefficient) with epigenetic pattern analysis, to identify CpG markers for body fluid identification. The selected CpGs were validated through pyrosequencing on peripheral blood, saliva, semen, vaginal secretions, and menstrual blood samples, and machine learning classification models were constructed based on the sequencing results. Pyrosequencing results revealed 14 CpGs with high specificity in five types of body fluid samples. A machine learning classification model, developed based on the pyrosequencing results, could effectively distinguish five types of body fluid samples, achieving 100 % accuracy on the test set. Utilizing six CpG markers, it was also feasible to attain ideal efficacy in identifying body fluid stains. Our research proposes a systematic and scientific strategy for screening body fluid-specific CpGs, contributing new insights and methods to forensic body fluid identification.
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Affiliation(s)
- Ming Zhao
- Guangzhou Key Laboratory of Forensic Multi-Omics for Precision Identification, School of Forensic Medicine, Southern Medical University, Guangzhou 510515, China
| | - Meiming Cai
- Guangzhou Key Laboratory of Forensic Multi-Omics for Precision Identification, School of Forensic Medicine, Southern Medical University, Guangzhou 510515, China
| | - Fanzhang Lei
- Guangzhou Key Laboratory of Forensic Multi-Omics for Precision Identification, School of Forensic Medicine, Southern Medical University, Guangzhou 510515, China
| | - Xi Yuan
- Guangzhou Key Laboratory of Forensic Multi-Omics for Precision Identification, School of Forensic Medicine, Southern Medical University, Guangzhou 510515, China
| | - Qinglin Liu
- Guangzhou Key Laboratory of Forensic Multi-Omics for Precision Identification, School of Forensic Medicine, Southern Medical University, Guangzhou 510515, China
| | - Yating Fang
- School of Basic Medical Science, Anhui Medical University, Hefei 230031, China.
| | - Bofeng Zhu
- Guangzhou Key Laboratory of Forensic Multi-Omics for Precision Identification, School of Forensic Medicine, Southern Medical University, Guangzhou 510515, China.
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Panda P, Mohapatra R. Revolutionizing DNA: advanced modification techniques for next-gen nanotechnology. NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS 2024:1-32. [PMID: 39589159 DOI: 10.1080/15257770.2024.2432992] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/29/2024] [Revised: 11/01/2024] [Accepted: 11/17/2024] [Indexed: 11/27/2024]
Abstract
The comprehensive advancement in DNA modification and coupling is driving DNA nanotechnology to new heights, paving the way for groundbreaking innovations in healthcare, materials science, and beyond. The ability to engineer DNA with tailored properties and functionalities underscores its immense potential in creating novel materials and devices. Utilizing a spectrum of techniques-such as amino handles, thiol groups, alkynes, azides, Diels-Alder reactions, hydrazides, and aminooxy functions-enables diverse coupling strategies, including Palladium-Catalyzed Couplings, to construct intricate DNA nanostructures. Further coupling modifications encompass hydrophobic alterations, redox-active moieties, chemical crosslinking agents, and Biotinylation. These modifications significantly broaden DNA's functional repertoire, offering precise control over interactions, structures, and features. By leveraging these advanced techniques, alongside next-generation sequencing (NGS)-based DNA modifications, researchers can design and implement DNA nanostructures with specific capabilities and applications, showcasing DNA's versatility as a programmable biomaterial. Through meticulous design and strategic implementation, DNA nanotechnology achieves unprecedented levels of precision and functionality, ushering in a new era of technological advancements and applications. These advanced DNA modification techniques hold great potential for transformative applications in nanotechnology, paving the way for innovations in drug delivery, diagnostics, and bioengineering.
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Affiliation(s)
- Pratikeswar Panda
- Department of Pharmaceutics, School of Pharmaceutical Science, Siksha 'O' Anusandhan University, Bhubaneswar, Odisha, India
| | - Rajaram Mohapatra
- Department of Pharmaceutics, School of Pharmaceutical Science, Siksha 'O' Anusandhan University, Bhubaneswar, Odisha, India
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4
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Xue L, Sun J, Sun Y, Wang Y, Zhang K, Fan M, Qian H, Li Y, Wang L. Maternal Brown Rice Diet during Pregnancy Promotes Adipose Tissue Browning in Offspring via Reprogramming PKA Signaling and DNA Methylation. Mol Nutr Food Res 2024:e2300861. [PMID: 38566521 DOI: 10.1002/mnfr.202300861] [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: 12/09/2023] [Revised: 02/23/2024] [Indexed: 04/04/2024]
Abstract
SCOPE Brown rice, the most consumed food worldwide, has been shown to possess beneficial effects on the prevention of metabolic diseases. However, the way in which maternal brown rice diet improves metabolism in offspring and the regulatory mechanisms remains unclear. The study explores the epigenetic regulation of offspring energy metabolic homeostasis by maternal brown rice diet during pregnancy. METHODS AND RESULTS Female mice are fed brown rice during pregnancy, and then body phenotypes, the histopathological analysis, and adipose tissues biochemistry assay of offspring mice are detected. It is found that maternal brown rice diet significantly reduces body weight and fat mass, increases energy expenditure and heat production in offspring. Maternal brown rice diet increases uncoupling protein 1 (UCP1) protein level and upregulates the mRNA expression of thermogenic genes in adipose tissues. Mechanistically, protein kinase A (PKA) signaling is likely responsible in the induced thermogenic program in offspring adipocytes, and the progeny adipocytes browning program is altered due to decreased level of DNA methyltransferase 1 protein and hypomethylation of the transcriptional coregulator positive regulatory domain containing 16 (PRDM16). CONCLUSIONS These findings demonstrate that maternal brown rice during pregnancy improves offspring mice metabolic homeostasis via promoting adipose browning, and its mechanisms may be mediated by DNA methylation reprogramming.
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Affiliation(s)
- Lamei Xue
- State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China
| | - Juan Sun
- State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China
| | - Yujie Sun
- State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China
| | - Yu Wang
- State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China
| | - Kuiliang Zhang
- State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China
| | - Mingcong Fan
- State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China
| | - Haifeng Qian
- State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China
| | - Yan Li
- State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China
| | - Li Wang
- State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China
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5
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Benn P, Cuckle H. Overview of Noninvasive Prenatal Testing (NIPT) for the Detection of Fetal Chromosome Abnormalities; Differences in Laboratory Methods and Scope of Testing. Clin Obstet Gynecol 2023; 66:536-556. [PMID: 37650667 DOI: 10.1097/grf.0000000000000803] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/01/2023]
Abstract
Although nearly all noninvasive prenatal testing is currently based on analyzing circulating maternal cell-free DNA, the technical methods usedvary considerably. We review the different methods. Based on validation trials and clinical experience, there are mostly relatively small differences in screening performance for trisomies 21, 18, and 13 in singleton pregnancies. Recent reports show low no-call rates for all methods, diminishing its importance when choosing a laboratory. However, method can be an important consideration for twin pregnancies, screening for sex chromosome abnormalities, microdeletion syndromes, triploidy, molar pregnancies, rare autosomal trisomies, and segmental imbalances, and detecting maternal chromosome abnormalities.
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Affiliation(s)
- Peter Benn
- Department of Obstetrics and Gynecology, University of Connecticut Health Center, Farmington, Connecticut
| | - Howard Cuckle
- Department of Obstetrics and Gynecology, Faculty of Medicine, Tel Aviv University, Israel
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6
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Alam A, Wilcox JE, Hall SA. The Traditional Endomyocardial Biopsy: Opportunities to Rethink Its Role as the Gold Standard. J Card Fail 2023; 29:1225-1227. [PMID: 34242781 DOI: 10.1016/j.cardfail.2021.05.029] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2021] [Revised: 05/16/2021] [Accepted: 05/18/2021] [Indexed: 11/18/2022]
Affiliation(s)
- Amit Alam
- Baylor University Medical Center, Dallas, Texas; Texas A&M University College of Medicine, Bryan, Texas.
| | - Jane E Wilcox
- Northwestern University Medical Center, Chicago, Illinois
| | - Shelley A Hall
- Baylor University Medical Center, Dallas, Texas; Texas A&M University College of Medicine, Bryan, Texas
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7
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Tian M, Feng L, Li J, Zhang R. Focus on the frontier issue: progress in noninvasive prenatal screening for fetal trisomy from clinical perspectives. Crit Rev Clin Lab Sci 2023; 60:248-269. [PMID: 36647189 DOI: 10.1080/10408363.2022.2162843] [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: 01/18/2023]
Abstract
The discovery of cell-free fetal DNA (cffDNA) in maternal blood and the rapid development of massively parallel sequencing have revolutionized prenatal testing from invasive to noninvasive. Noninvasive prenatal screening (NIPS) based on cffDNA enables the detection of fetal trisomy through sequencing, comparison, and bioassays. Its accuracy is better than that of traditional screening methods, and it is the most advanced clinical application of high-throughput sequencing technologies. However, the existing sequencing methods are limited by high costs and complex sequencing procedures. These limitations restrict the availability of NIPS for pregnant women. Many amplification methods have been developed to overcome the limitations of sequencing methods. The rapid development of non-sequencing methods has not been accompanied by reviews to summarize them. In this review, we initially describe the detection principles for sequencing-based NIPS. We summarize the rapidly evolving amplification technologies, focusing on the need to reduce costs and simplify the procedures. To ensure that the testing systems are feasible and that the testing processes are reliable, we expand our vision to the clinic. We evaluate the clinical validity of NIPS in terms of sensitivity, specificity, and positive predictive value. Finally, we summarize the application guidelines and discuss the corresponding quality control methods for NIPS. In addition to cffDNA, extracellular vesicle DNA, RNA, protein/peptide, and fetal cells can also be detected as biomarkers of NIPS. With the development of prenatal testing, NIPS has become increasingly important. Notably, NIPS is a screening test instead of a diagnostic test. The testing methods and procedures used in the NIPS process require standardization.
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Affiliation(s)
- Meng Tian
- National Center for Clinical Laboratories, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing Hospital/National Center of Gerontology, Beijing, P. R. China.,Peking University Fifth School of Clinical Medicine, Beijing, P. R. China.,Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, P. R. China
| | - Lei Feng
- National Center for Clinical Laboratories, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing Hospital/National Center of Gerontology, Beijing, P. R. China.,Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, P. R. China.,Graduate School of Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, P. R. China
| | - Jinming Li
- National Center for Clinical Laboratories, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing Hospital/National Center of Gerontology, Beijing, P. R. China.,Peking University Fifth School of Clinical Medicine, Beijing, P. R. China.,Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, P. R. China.,Graduate School of Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, P. R. China
| | - Rui Zhang
- National Center for Clinical Laboratories, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing Hospital/National Center of Gerontology, Beijing, P. R. China.,Peking University Fifth School of Clinical Medicine, Beijing, P. R. China.,Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, P. R. China.,Graduate School of Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, P. R. China
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8
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Műzes G, Bohusné Barta B, Szabó O, Horgas V, Sipos F. Cell-Free DNA in the Pathogenesis and Therapy of Non-Infectious Inflammations and Tumors. Biomedicines 2022; 10:biomedicines10112853. [PMID: 36359370 PMCID: PMC9687442 DOI: 10.3390/biomedicines10112853] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2022] [Revised: 10/31/2022] [Accepted: 11/07/2022] [Indexed: 11/09/2022] Open
Abstract
The basic function of the immune system is the protection of the host against infections, along with the preservation of the individual antigenic identity. The process of self-tolerance covers the discrimination between self and foreign antigens, including proteins, nucleic acids, and larger molecules. Consequently, a broken immunological self-tolerance results in the development of autoimmune or autoinflammatory disorders. Immunocompetent cells express pattern-recognition receptors on their cell membrane and cytoplasm. The majority of endogenous DNA is located intracellularly within nuclei and mitochondria. However, extracellular, cell-free DNA (cfDNA) can also be detected in a variety of diseases, such as autoimmune disorders and malignancies, which has sparked interest in using cfDNA as a possible biomarker. In recent years, the widespread use of liquid biopsies and the increasing demand for screening, as well as monitoring disease activity and therapy response, have enabled the revival of cfDNA research. The majority of studies have mainly focused on the function of cfDNA as a biomarker. However, research regarding the immunological consequences of cfDNA, such as its potential immunomodulatory or therapeutic benefits, is still in its infancy. This article discusses the involvement of various DNA-sensing receptors (e.g., absent in melanoma-2; Toll-like receptor 9; cyclic GMP-AMP synthase/activator of interferon genes) in identifying host cfDNA as a potent danger-associated molecular pattern. Furthermore, we aim to summarize the results of the experimental studies that we recently performed and highlight the immunomodulatory capacity of cfDNA, and thus, the potential for possible therapeutic consideration.
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Affiliation(s)
| | | | | | | | - Ferenc Sipos
- Correspondence: ; Tel.: +36-20-478-0752; Fax: +36-1-266-0816
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Xue L, Sun J, Liu J, Hu C, Wu D, Nie C, Zhang K, Wang Y, Zhao L, Li X, Lu Y, Zhang L, Zhang D, Fan M, Qian H, Jiang H, Wong J, Li Y, Ying H, Chow BKC, Wang L, Li Y. Maternal secretin ameliorates obesity by promoting white adipose tissue browning in offspring. EMBO Rep 2022; 23:e54132. [PMID: 35652247 PMCID: PMC9253765 DOI: 10.15252/embr.202154132] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2021] [Revised: 04/26/2022] [Accepted: 05/05/2022] [Indexed: 12/09/2023] Open
Abstract
Our knowledge of the coordination of intergenerational inheritance and offspring metabolic reprogramming by gastrointestinal endocrine factors is largely unknown. Here, we showed that secretin (SCT), a brain-gut peptide, is downregulated by overnutrition in pregnant mice and women. More importantly, genetic loss of SCT in the maternal gut results in undesirable phenotypes developed in offspring including enhanced high-fat diet (HFD)-induced obesity and attenuated browning of inguinal white adipose tissue (iWAT). Mechanistically, loss of maternal SCT represses iWAT browning in offspring by a global change in genome methylation pattern through upregulation of DNMT1. SCT functions to facilitate ubiquitination and degradation of DNMT1 by activating AMPKα, which contributes to the observed alteration of DNMT1 in progeny. Lastly, we showed that SCT treatment during pregnancy can reduce the development of obesity and improve glucose tolerance and insulin resistance in offspring of HFD-fed females, suggesting that SCT may serve as a novel biomarker or a strategy for preventing metabolic diseases.
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Affiliation(s)
- Lamei Xue
- State Key Laboratory of Food Science and TechnologySchool of Food Science and TechnologyJiangnan UniversityWuxiChina
| | - Juan Sun
- State Key Laboratory of Food Science and TechnologySchool of Food Science and TechnologyJiangnan UniversityWuxiChina
| | - Jinxin Liu
- State Key Laboratory of Food Science and TechnologySchool of Food Science and TechnologyJiangnan UniversityWuxiChina
| | - Chaoping Hu
- Department of Neuromuscular DiseaseChildren’s Hospital of Fudan UniversityShanghaiChina
| | - Dandan Wu
- Shanghai Key Laboratory of StomatologyDepartment of Oral & Cranio‐maxillofacial ScienceShanghai 9th People's HospitalCollege of StomatologySchool of MedicineShanghai Jiao Tong UniversityShanghaiChina
| | - Chenzhipeng Nie
- State Key Laboratory of Food Science and TechnologySchool of Food Science and TechnologyJiangnan UniversityWuxiChina
| | - Kuiliang Zhang
- State Key Laboratory of Food Science and TechnologySchool of Food Science and TechnologyJiangnan UniversityWuxiChina
| | - Yu Wang
- State Key Laboratory of Food Science and TechnologySchool of Food Science and TechnologyJiangnan UniversityWuxiChina
| | - Lei Zhao
- Department of Neuromuscular DiseaseChildren’s Hospital of Fudan UniversityShanghaiChina
| | - Xihua Li
- Department of Neuromuscular DiseaseChildren’s Hospital of Fudan UniversityShanghaiChina
| | - Yan Lu
- Department of Endocrinology and MetabolismZhongshan HospitalFudan UniversityShanghaiChina
| | - Li Zhang
- Joint International Research Laboratory of CNS RegenerationGuangdong‐Hong Kong‐Macau Institute of CNS RegenerationJinan UniversityGuangzhouChina
| | - Duo Zhang
- Clinical and Experimental TherapeuticsCollege of PharmacyUniversity of Georgia and Charlie Norwood VA Medical CenterAugustaGAUSA
| | - Mingcong Fan
- State Key Laboratory of Food Science and TechnologySchool of Food Science and TechnologyJiangnan UniversityWuxiChina
| | - Haifeng Qian
- State Key Laboratory of Food Science and TechnologySchool of Food Science and TechnologyJiangnan UniversityWuxiChina
| | - Haowen Jiang
- State Key Laboratory of Drug ResearchShanghai Institute of Materia MedicaChinese Academy of SciencesShanghaiChina
| | - Jiemin Wong
- Shanghai Key Laboratory of Regulatory BiologyFengxian District Central Hospital‐ECNU Joint Center of Translational MedicineInstitute of Biomedical Sciences and School of Life SciencesEast China Normal UniversityShanghaiChina
| | - Yuying Li
- Chinese Academy of Sciences Key Laboratory of Nutrition, Metabolism and Food SafetyShanghai Institutes for Biological SciencesChinese Academy of SciencesShanghaiChina
| | - Hao Ying
- Chinese Academy of Sciences Key Laboratory of Nutrition, Metabolism and Food SafetyShanghai Institutes for Biological SciencesChinese Academy of SciencesShanghaiChina
| | - Billy KC Chow
- School of Biological SciencesUniversity of Hong KongHong KongChina
| | - Li Wang
- State Key Laboratory of Food Science and TechnologySchool of Food Science and TechnologyJiangnan UniversityWuxiChina
| | - Yan Li
- State Key Laboratory of Food Science and TechnologySchool of Food Science and TechnologyJiangnan UniversityWuxiChina
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10
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Ochoa E, Zuber V, Bottolo L. Accurate Measurement of DNA Methylation: Challenges and Bias Correction. Methods Mol Biol 2022; 2432:25-47. [PMID: 35505205 DOI: 10.1007/978-1-0716-1994-0_3] [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] [Indexed: 06/14/2023]
Abstract
DNA methylation is a key epigenetic modification involved in gene regulation whose contribution to disease susceptibility is still not fully understood. As the cost of genome sequencing technologies continues to drop, it will soon become commonplace to perform genome-wide quantification of DNA methylation at a single base-pair resolution. However, the demand for its accurate quantification might vary across studies. When the scope of the analysis is to detect regions of the genome with different methylation patterns between two or more conditions, e.g., case vs control; treatments vs placebo, accuracy is not crucial. This is the case in epigenome-wide association studies used as genome-wide screening of methylation changes to detect new candidate genes and regions associated with a specific disease or condition. If the aim of the analysis is to use DNA methylation measurements as a biomarker for diseases diagnosis and treatment (Laird, Nat Rev Cancer 3:253-266, 2003; Bock, Epigenomics 1:99-110, 2009), it is instead recommended to produce accurate methylation measurements. Furthermore, if the objective is the detection of DNA methylation in subclonal tumor cell populations or in circulating tumor DNA or in any case of mosaicism, the importance of accuracy becomes critical. The aim of this chapter is to describe the factors that could affect the precise measurement of methylation levels and a recent Bayesian statistical method called MethylCal and its extension that have been proposed to minimize this problem.
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Affiliation(s)
- Eguzkine Ochoa
- Department of Medical Genetics, University of Cambridge, Cambridge, UK
- Cambridge NIHR Biomedical Research Centre, Cambridge, UK
| | - Verena Zuber
- Department of Epidemiology and Biostatistics, Imperial College London, London, UK
- MRC Biostatistics Unit, University of Cambridge, Cambridge, UK
| | - Leonardo Bottolo
- Department of Medical Genetics, University of Cambridge, Cambridge, UK.
- MRC Biostatistics Unit, University of Cambridge, Cambridge, UK.
- The Alan Turing Institute, London, UK.
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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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12
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Sharma M, Verma RK, Kumar S, Kumar V. Computational challenges in detection of cancer using cell-free DNA methylation. Comput Struct Biotechnol J 2021; 20:26-39. [PMID: 34976309 PMCID: PMC8669313 DOI: 10.1016/j.csbj.2021.12.001] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2021] [Revised: 12/02/2021] [Accepted: 12/02/2021] [Indexed: 12/18/2022] Open
Abstract
Cell-free DNA(cfDNA) methylation profiling is considered promising and potentially reliable for liquid biopsy to study progress of diseases and develop reliable and consistent diagnostic and prognostic biomarkers. There are several different mechanisms responsible for the release of cfDNA in blood plasma, and henceforth it can provide information regarding dynamic changes in the human body. Due to the fragmented nature, low concentration of cfDNA, and high background noise, there are several challenges in its analysis for regular use in diagnosis of cancer. Such challenges in the analysis of the methylation profile of cfDNA are further aggravated due to heterogeneity, biomarker sensitivity, platform biases, and batch effects. This review delineates the origin of cfDNA methylation, its profiling, and associated computational problems in analysis for diagnosis. Here we also contemplate upon the multi-marker approach to handle the scenario of cancer heterogeneity and explore the utility of markers for 5hmC based cfDNA methylation pattern. Further, we provide a critical overview of deconvolution and machine learning methods for cfDNA methylation analysis. Our review of current methods reveals the potential for further improvement in analysis strategies for detecting early cancer using cfDNA methylation.
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Key Words
- Cancer heterogeneity
- Cell free DNA
- Computation
- DMP, Differentially methylated base position
- DMR, Differentially methylated regions
- Diagnosis
- HELP-seq, HpaII-tiny fragment Enrichment by Ligation-mediated PCR sequencing
- MBD-seq, Methyl-CpG Binding Domain Protein Capture Sequencing
- MCTA-seq, Methylated CpG tandems amplification and sequencing
- MSCC, Methylation Sensitive Cut Counting
- MSRE, methylation sensitive restriction enzymes
- MeDIP-seq, Methylated DNA Immunoprecipitation Sequencing
- RRBS, Reduced-Representation Bisulfite Sequencing
- WGBS, Whole Genome Bisulfite Sequencing
- cfDNA, cell free DNA
- ctDNA, circulating tumor DNA
- dPCR, digital polymerase chain reaction
- ddMCP, droplet digital methylation-specific PCR
- ddPCR, droplet digital polymerase chain reaction
- scCGI, methylated CGIs at single cell level
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Affiliation(s)
- Madhu Sharma
- Department for Computational Biology, Indraprastha Institute of Information Technology, Delhi 110020, India
| | - Rohit Kumar Verma
- Department for Computational Biology, Indraprastha Institute of Information Technology, Delhi 110020, India
| | - Sunil Kumar
- Department of Surgical oncology, All India Institute of Medical sciences, New Delhi 110029, India
| | - Vibhor Kumar
- Department for Computational Biology, Indraprastha Institute of Information Technology, Delhi 110020, India
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Martínez-Sánchez N, Robles Marhuenda A, De la Calle Fernández-Miranda M, Bartha JL. First trimester combined screening test for aneuploidies in anti-Ro carriers pregnant women. Clin Rheumatol 2021; 40:2699-2705. [PMID: 33559011 DOI: 10.1007/s10067-021-05616-8] [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: 05/02/2020] [Revised: 01/22/2021] [Accepted: 01/26/2021] [Indexed: 12/01/2022]
Abstract
INTRODUCTION Anti-Ro/SSA and anti-La/SSB antibodies are associated with neonatal lupus and congenital heart block. Controversial results regarding perinatal outcomes are found and less is known about aneuploidy screening. The hypothesis is that the presence of anti-Ro and/or anti-La antibodies influences the levels of PAPP-A and ß-HCG, thus interfering in the calculation of risk of aneuploidies. MATERIAL AND METHODS Fifty-five anti-Ro/SSA positive pregnant women were included. The demographic characteristics and laboratory variables were studied. Data concerning chromosomopaties screening were also recorded. RESULTS PAPP-A and β-HCG levels were calculated (as well as NT and CRL) and compared with a healthy cohort of 12971 pregnant women. PAPP-A levels in mg/mL were lower significatively. In anti-La/SS-B cohort, significant differences were found in PAPP-A in mg/mL and in MoM. Combined risks for Down syndrome (DS) in both groups were higher but the differences were due to age. CONCLUSIONS Serum levels of PAPP-A were significative lower but not confirmed when adjusted to MoM. This will have to be confirmed in studies with a larger number of patients and to check whether there is an impact in the calculation of DS risk or not. They could represent a group of pregnant women with significantly a higher risk of adverse perinatal outcome. Key Points • Pregnant patients with anti-Ro/SS-A ant/or anti-La/SS-B antibodies have low PAPP-A levels compared with pregnant women without antibodies. • PAPP-A levels are used in obstetrics for aneuploidies screening in the first trimester, so in these patients, there could be more false positive screening. • In these findings are verified in trials with a larger number of patients, a correction variable would have to be applied for the aneuploidies screening calculation. • Also, low PAPP-A levels are correlated with poor placentation, that is to say, more risk of miscarriages, small fetus for gestational age, and preeclampsia. This is another topic to take into consideration in this population.
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Affiliation(s)
- N Martínez-Sánchez
- Obstetrics and Gynecology Department, University Hospital La Paz, Madrid, Spain.
| | | | | | - J L Bartha
- Obstetrics and Gynecology Department, University Hospital La Paz, Madrid, Spain
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Blais J, Giroux S, Caron A, Clément V, Rousseau F. Precision of Fetal DNA Fraction Estimation by Quantitative Polymerase Chain Reaction Quantification of a Differently Methylated Target in Noninvasive Prenatal Testing. Lab Med 2020; 51:279-287. [PMID: 31755528 DOI: 10.1093/labmed/lmz068] [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: 11/14/2022] Open
Abstract
BACKGROUND The performance of noninvasive prenatal testing (NIPT) assays is critically determined by the proportion of fetal DNA or fetal fraction (FF). Fetomaternal differential methylation of certain genomic regions has been proposed as a universal marker of fetal origin, and previous reports have suggested the use of methylation-sensitive restriction enzyme (MSRE) assays to estimate FF. METHODS We analyzed the performance of FF estimation using an MSRE assay with duplex quantitative polymerase chain reaction (qPCR). Mixtures of genomic DNA from placental cells and from adult women were digested with 2 MSRE and FF estimates obtained, for a total of 221 pairwise treatment/control comparisons. RESULTS The coefficient of variance (CV) of the MSRE assays was high, ranging from 24% to 60%. An alternative in silico FF estimation algorithm, SeqFF, displayed slightly lower variability, with a CV of 22%. CONCLUSION These results cast doubts on the usefulness of the MSRE-based assay of differentially methylated markers for FF estimation. The lack of a universal method capable of precisely estimating FF remains an incompletely solved issue.
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Affiliation(s)
- Jonatan Blais
- Medical Biochemistry Service, Department of Laboratory Medicine, CHU (Centre hospitalier universitaire) de Québec-Université Laval, Quebec City, Quebec, Canada.,Department of Molecular Biology, Medical Biochemistry, and Pathology, Faculty of Medicine, Université Laval, Quebec City, Quebec, Canada.,Human and Molecular Genetics Research Unit, Research Center, CHU de Québec, Quebec City, Quebec, Canada
| | - Sylvie Giroux
- Human and Molecular Genetics Research Unit, Research Center, CHU de Québec, Quebec City, Quebec, Canada
| | - André Caron
- Human and Molecular Genetics Research Unit, Research Center, CHU de Québec, Quebec City, Quebec, Canada
| | - Valérie Clément
- Human and Molecular Genetics Research Unit, Research Center, CHU de Québec, Quebec City, Quebec, Canada
| | - François Rousseau
- Medical Biochemistry Service, Department of Laboratory Medicine, CHU (Centre hospitalier universitaire) de Québec-Université Laval, Quebec City, Quebec, Canada.,Department of Molecular Biology, Medical Biochemistry, and Pathology, Faculty of Medicine, Université Laval, Quebec City, Quebec, Canada.,Human and Molecular Genetics Research Unit, Research Center, CHU de Québec, Quebec City, Quebec, Canada.,PEGASUS (Personalized Genomics for Prenatal Abnormalities Screening Using Maternal Blood), Quebec City, Quebec, Canada
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15
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Gordevičius J, Narmontė M, Gibas P, Kvederavičiūtė K, Tomkutė V, Paluoja P, Krjutškov K, Salumets A, Kriukienė E. Identification of fetal unmodified and 5-hydroxymethylated CG sites in maternal cell-free DNA for non-invasive prenatal testing. Clin Epigenetics 2020; 12:153. [PMID: 33081811 PMCID: PMC7574562 DOI: 10.1186/s13148-020-00938-x] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2020] [Accepted: 09/14/2020] [Indexed: 12/14/2022] Open
Abstract
BACKGROUND Massively parallel sequencing of maternal cell-free DNA (cfDNA) is widely used to test fetal genetic abnormalities in non-invasive prenatal testing (NIPT). However, sequencing-based approaches are still of high cost. Building upon previous knowledge that placenta, the main source of fetal circulating DNA, is hypomethylated in comparison to maternal tissue counterparts of cfDNA, we propose that targeting either unmodified or 5-hydroxymethylated CG sites specifically enriches fetal genetic material and reduces numbers of required analytical sequencing reads thereby decreasing cost of a test. METHODS We employed uTOPseq and hmTOP-seq approaches which combine covalent derivatization of unmodified or hydroxymethylated CG sites, respectively, with next generation sequencing, or quantitative real-time PCR. RESULTS We detected increased 5-hydroxymethylcytosine (5hmC) levels in fetal chorionic villi (CV) tissue samples as compared with peripheral blood. Using our previously developed uTOP-seq and hmTOP-seq approaches we obtained whole-genome uCG and 5hmCG maps of 10 CV tissue and 38 cfDNA samples in total. Our results indicated that, in contrast to conventional whole genome sequencing, such epigenomic analysis highly specifically enriches fetal DNA fragments from maternal cfDNA. While both our approaches yielded 100% accuracy in detecting Down syndrome in fetuses, hmTOP-seq maintained such accuracy at ultra-low sequencing depths using only one million reads. We identified 2164 and 1589 placenta-specific differentially modified and 5-hydroxymethylated regions, respectively, in chromosome 21, as well as 3490 and 2002 Down syndrome-specific differentially modified and 5-hydroxymethylated regions, respectively, that can be used as biomarkers for identification of Down syndrome or other epigenetic diseases of a fetus. CONCLUSIONS uTOP-seq and hmTOP-seq approaches provide a cost-efficient and sensitive epigenetic analysis of fetal abnormalities in maternal cfDNA. The results demonstrated that T21 fetuses contain a perturbed epigenome and also indicated that fetal cfDNA might originate from fetal tissues other than placental chorionic villi. Robust covalent derivatization followed by targeted analysis of fetal DNA by sequencing or qPCR presents an attractive strategy that could help achieve superior sensitivity and specificity in prenatal diagnostics.
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Affiliation(s)
- Juozas Gordevičius
- Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, 10257, Vilnius, Lithuania
| | - Milda Narmontė
- Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, 10257, Vilnius, Lithuania
| | - Povilas Gibas
- Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, 10257, Vilnius, Lithuania
| | - Kotryna Kvederavičiūtė
- Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, 10257, Vilnius, Lithuania
| | - Vita Tomkutė
- Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, 10257, Vilnius, Lithuania
| | - Priit Paluoja
- Competence Centre On Health Technologies, Teaduspargi 13, 50411, Tartu, Estonia.,Faculty of Medicine, University of Helsinki, 00014, Helsinki, Finland
| | - Kaarel Krjutškov
- Competence Centre On Health Technologies, Teaduspargi 13, 50411, Tartu, Estonia.,Department of Obstetrics and Gynaecology, Institute of Clinical Medicine, University of Tartu, L. Puusepa 8, 50406, Tartu, Estonia
| | - Andres Salumets
- Competence Centre On Health Technologies, Teaduspargi 13, 50411, Tartu, Estonia.,Department of Obstetrics and Gynaecology, Institute of Clinical Medicine, University of Tartu, L. Puusepa 8, 50406, Tartu, Estonia.,Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, HUS, PO Box 140, 00029, Helsinki, Finland.,Estonian Genome Center, Institute of Genomics, University of Tartu, Riia 23b, 51010, Tartu, Estonia
| | - Edita Kriukienė
- Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, 10257, Vilnius, Lithuania. .,Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, 10257, Vilnius, Lithuania.
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16
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Peng X, Li HD, Wu FX, Wang J. Identifying the tissues-of-origin of circulating cell-free DNAs is a promising way in noninvasive diagnostics. Brief Bioinform 2020; 22:5840077. [PMID: 32427285 DOI: 10.1093/bib/bbaa060] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2020] [Revised: 03/16/2020] [Accepted: 03/25/2020] [Indexed: 12/18/2022] Open
Abstract
Advances in sequencing technologies facilitate personalized disease-risk profiling and clinical diagnosis. In recent years, some great progress has been made in noninvasive diagnoses based on cell-free DNAs (cfDNAs). It exploits the fact that dead cells release DNA fragments into the circulation, and some DNA fragments carry information that indicates their tissues-of-origin (TOOs). Based on the signals used for identifying the TOOs of cfDNAs, the existing methods can be classified into three categories: cfDNA mutation-based methods, methylation pattern-based methods and cfDNA fragmentation pattern-based methods. In cfDNA mutation-based methods, the SNP information or the detected mutations in driven genes of certain diseases are employed to identify the TOOs of cfDNAs. Methylation pattern-based methods are developed to identify the TOOs of cfDNAs based on the tissue-specific methylation patterns. In cfDNA fragmentation pattern-based methods, cfDNA fragmentation patterns, such as nucleosome positioning or preferred end coordinates of cfDNAs, are used to predict the TOOs of cfDNAs. In this paper, the strategies and challenges in each category are reviewed. Furthermore, the representative applications based on the TOOs of cfDNAs, including noninvasive prenatal testing, noninvasive cancer screening, transplantation rejection monitoring and parasitic infection detection, are also reviewed. Moreover, the challenges and future work in identifying the TOOs of cfDNAs are discussed. Our research provides a comprehensive picture of the development and challenges in identifying the TOOs of cfDNAs, which may benefit bioinformatics researchers to develop new methods to improve the identification of the TOOs of cfDNAs.
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Ioannides M, Achilleos A, Kyriakou S, Kypri E, Loizides C, Tsangaras K, Constantinou L, Koumbaris G, Patsalis PC. Development of a new methylation-based fetal fraction estimation assay using multiplex ddPCR. Mol Genet Genomic Med 2019; 8:e1094. [PMID: 31821748 PMCID: PMC7005606 DOI: 10.1002/mgg3.1094] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2019] [Accepted: 11/17/2019] [Indexed: 01/12/2023] Open
Abstract
Background Non‐invasive prenatal testing (NIPT) for fetal aneuploidies has rapidly been incorporated into clinical practice. Current NGS‐based methods can reliably detect fetal aneuploidies non‐invasively with fetal fraction of at least 4%. Inaccurate fetal fraction assessment can compromise the accuracy of the test as affected samples with low fetal fraction have an increased risk for misdiagnosis. Using a novel set of fetal‐specific differentially methylated regions (DMRs) and methylation sensitive restriction digestion (MSRD), we developed a multiplex ddPCR assay for accurate detection of fetal fraction in maternal plasma. Methods We initially performed MSRD followed by methylation DNA immunoprecipitation (MeDIP) and NGS on fetal and non‐pregnant female tissues to identify fetal‐specific DMRs. DMRs with the highest methylation difference between the two tissues were selected for fetal fraction estimation employing MSRD and multiplex ddPCR. Chromosome Y multiplex ddPCR assay (YMM) was used as a reference standard, to develop our fetal fraction estimation model in male pregnancy samples. Additional 123 samples were tested to examine whether the model is sex dependent and/or ploidy dependent. Results In all, 93 DMRs were identified of which seven were selected for fetal fraction estimation. Statistical analysis resulted in the final model which included four DMRs (FFMM). High correlation with YMM‐based fetal fractions was observed using 85 male pregnancies (r = 0.86 95% CI: 0.80–0.91). The model was confirmed using an independent set of 53 male pregnancies. Conclusion By employing a set of well‐characterized DMRs, we developed a SNP‐, sex‐ and ploidy‐independent methylation‐based multiplex ddPCR assay for accurate fetal fraction estimation.
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18
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Kumar N, Singh AK. Cell-Free Fetal DNA: A Novel Biomarker for Early Prediction of Pre-eclampsia and Other Obstetric Complications. Curr Hypertens Rev 2019; 15:57-63. [PMID: 29766818 DOI: 10.2174/1573402114666180516131832] [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: 01/10/2018] [Revised: 05/06/2018] [Accepted: 05/11/2018] [Indexed: 11/22/2022]
Abstract
Hypertensive disorder of pregnancy, especially Pre-eclampsia is one of the major causes of increased maternal and perinatal morbidity and mortality all over the world. Early prediction of pre-eclampsia is the need of modern obstetrics, as this can timely prevent the progress of disease as well as related fetal and maternal morbidity and mortality. In addition to the screening of fetal aneuploidies, Rhesus-D status, fetal sex, single gene disorders, the cell-free fetal Deoxyribonucleic acid (DNA) quantification has emerged as a promising biomarker for the prediction of pre-eclampsia. Hence, its use can help in the early prediction of hypertensive disorders of pregnancy, especially pre-eclampsia even before the appearance of symptoms. Furthermore, in future, it can also help in the determination of the complete DNA sequence of every gene of the fetus. The present review focuses on recent literature concerning the use of cell-free fetal DNA in early prediction of preeclampsia as well as for non-invasive prenatal genetic screening of fetus for various disorders. Methods: The recent literature related to cell-free fetal DNA was searched from numerous English language journals and published peer-reviewed articles on Pubmed, Google Scholar, MEDLINE and various government agencies till 2016.
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Affiliation(s)
- Naina Kumar
- Department of Obstetrics and Gynecology, Maharishi Markandeshwar Institute of Medical Sciences and Research, Mullana-133207, Ambala, Haryana, India, Postal address: House Number 2, "F" Block, MMIMSR Campus, Mullana- 133207, Ambala, Haryana, India
| | - Amit Kant Singh
- Department of Physiology, U.P. University of Medical Sciences, Saifai, Etawah, Uttar Pradesh, India, Postal Address: House No. 168, Kaveri Kunj, Phase II, Kamla Nagar-282005, Agra, Uttar Pradesh, India
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19
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Zhang R, Yin Y, Zhang S, Chen L, Pu L, Deng Q, Zhang H, Xiao L. Application of Differentially Methylated Loci in Clinical Diagnosis of Trisomy 21 Syndrome. Genet Test Mol Biomarkers 2019; 23:246-250. [PMID: 30986102 DOI: 10.1089/gtmb.2018.0176] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
AIMS To determine the diagnostic precision of using different sets of fetal-specific methylation markers with methylation-sensitive restriction enzyme-quantitative polymerase chain reaction (MSRE-qPCR) for detection of trisomy 21 (T21). MATERIALS AND METHODS The diagnostic value for trisomy 21 of differential methylation of HLCS, C21orf25, and RASSF1A (a fetal-specific internal control) was examined by MSRE-qPCR. RESULTS The combined marker set of HLCS and RASSF1A achieved accurate quantification of fetal-specific chromosome 21 and was an excellent marker for detecting the presence of three copies of chromosome 21. MSRE-qPCR correctly identified three cases of fetal T21 from 11 clinical samples, which were 100% consistent with karyotyping results. In addition, this method was able to detect fetal-specific, T21-derived, cell-free fetal DNA at concentrations as low as 0.1%. CONCLUSIONS Evaluation of the HLCS and RASSF1A fetal-specific methylation marker set by MSRE-qPCR could be a highly sensitive, specific, cost-effective, and noninvasive prenatal screening method for T21. This MSRE-qPCR testable marker should be considered as an alternative to next generation sequencing technology for diagnosing fetal T21.
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Affiliation(s)
- Rong Zhang
- 1 Department of Obstetrics and Gynecology, Second Affiliated Hospital of Soochow University, Suzhou, China
| | - Yufang Yin
- 2 Department of Pharmacology and Neuroscience, School of Medicine, Southern Illinois University, Springfield, Illinois
| | - Shuyun Zhang
- 1 Department of Obstetrics and Gynecology, Second Affiliated Hospital of Soochow University, Suzhou, China
| | - Li Chen
- 1 Department of Obstetrics and Gynecology, Second Affiliated Hospital of Soochow University, Suzhou, China
| | - Li Pu
- 1 Department of Obstetrics and Gynecology, Second Affiliated Hospital of Soochow University, Suzhou, China
| | - Qicheng Deng
- 1 Department of Obstetrics and Gynecology, Second Affiliated Hospital of Soochow University, Suzhou, China
| | - Hong Zhang
- 1 Department of Obstetrics and Gynecology, Second Affiliated Hospital of Soochow University, Suzhou, China
| | - Li Xiao
- 3 Department of Molecular Medicine Center, Second Affiliated Hospital of Soochow University, Suzhou, China
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20
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Abstract
Since its discovery in human blood plasma about 70 years ago, circulating cell-free DNA (cfDNA) has become an attractive subject of research as noninvasive disease biomarker. The interest in clinical applications has gained an exponential increase, making it a popular and potential target in a wide range of research areas.cfDNA can be found in different body fluids, both in healthy and not healthy subjects. The recent and rapid development of new molecular techniques is promoting the study and the identification of cfDNA, holding the key to minimally invasive diagnostics, improving disease monitoring, clinical decision, and patients' outcome.cfDNA has already given a huge impact on prenatal medicine, and it could become, in the next future, the standard of care also in other fields, from oncology to transplant medicine and cardiovascular diseases.
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Affiliation(s)
- Rossella Ranucci
- Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy.
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21
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Abstract
During the past decades, life expectancy of subjects with Down syndrome (DS) has greatly improved, but age-specific mortality rates are still important and DS subjects are characterized by an acceleration of the ageing process, which affects particularly the immune and central nervous systems. In this chapter, we will first review the characteristics of the ageing phenomenon in brain and in immune system in DS and we will then discuss the biological hallmarks of ageing in this specific population. Finally, we will also consider in detail the knowledge on epigenetics in DS, particularly DNA methylation.
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Abstract
Prenatal testing in recent years has been moving toward non-invasive methods to determine the fetal risk for genetic disorders without incurring the risk of miscarriage. Rapid progress of modern high-throughput molecular technologies along with the discovery of cell-free fetal DNA in maternal plasma led to novel screening methods for fetal chromosomal aneuploidies. Such tests are referred to as non-invasive prenatal tests (NIPTs), non-invasive prenatal screening, or prenatal cell-free DNA screening. Owing to many advantages, the adoption of NIPT in routine clinical practice was very rapid and global. As an example, NIPT has recently become a standard screening procedure for all pregnant women in the Netherlands. On the other hand, invasive sampling procedures remain important, especially for their diagnostic value in the confirmation of NIPT-positive findings and the detection of Mendelian disorders. In this review, we focus on current trends in the field of NIPT and discuss their benefits, drawbacks, and consequences in regard to routine diagnostics.
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Affiliation(s)
- Ondrej Pös
- Faculty of Natural Sciences, Comenius University, Bratislava, 84215, Slovakia
| | - Jaroslav Budiš
- University Science Park, Comenius University, Bratislava, 84104, Slovakia
| | - Tomáš Szemes
- Faculty of Natural Sciences, Comenius University, Bratislava, 84215, Slovakia.,University Science Park, Comenius University, Bratislava, 84104, Slovakia
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Chen H, Ji M, Zong JF, Ko JMY, Dai W, Lung ML. Conventional and Novel Diagnostic Biomarkers and Approaches for Detection of Nasopharyngeal Carcinoma. NASOPHARYNGEAL CARCINOMA 2019:129-153. [DOI: 10.1016/b978-0-12-814936-2.00007-9] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
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Lim JH, Lee BY, Kim JW, Han YJ, Kim MH, Chung JH, Han JY, Kim MY, Ryu HM. Effective Fetal Epigenetic Biomarkers for Noninvasive Fetal Trisomy 21 Detections. Fetal Diagn Ther 2018; 46:133-138. [PMID: 30408793 DOI: 10.1159/000494054] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2018] [Accepted: 09/25/2018] [Indexed: 11/19/2022]
Abstract
INTRODUCTION Recently, we identified three novel fetal-specific epigenetic DNA regions (FSERs) on chromosome 21 for detection of noninvasive fetal trisomy 21 (T21). In this study, the diagnostic accuracies of the three FSERs were assessed on a larger panel of the first-trimester pregnant women. MATERIAL AND METHODS This study was conducted with maternal plasma collected from 167 pregnant women carrying 155 chromosomally normal and 12 T21 fetuses (10-13 gestational weeks). Accuracies of FSERs for noninvasive prenatal test of fetal T21 were estimated by the area under the receiver operator characteristic curve (AUC). RESULTS The levels of all FSERs increased in pregnant women with T21 fetuses when compared with controls (p < 0.001 for all). The levels of the three FSERs did not differ according to maternal age, body mass index, and fetal sex at maternal blood sampling (p > 0.05 for all). In noninvasive fetal T21 detection, the AUC of FSER1, FSER2, and FSER3 were 0.859 (95% CI: 0.746-0.972), 0.919 (95% CI: 0.856-0.982), and 0.868 (95% CI: 0.746-0.990), respectively. DISCUSSION The findings of this study suggest that all FSERs may be useful for noninvasive fetal T21 detection, regardless of maternal age, body mass index, and fetal sex.
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Affiliation(s)
- Ji Hyae Lim
- Laboratory of Medical Genetics, Medical Research Institute, Cheil General Hospital and Women's Healthcare Center, Seoul, Republic of Korea
| | - Bom Yi Lee
- Laboratory of Medical Genetics, Medical Research Institute, Cheil General Hospital and Women's Healthcare Center, Seoul, Republic of Korea
| | - Jin Woo Kim
- Laboratory of Medical Genetics, Medical Research Institute, Cheil General Hospital and Women's Healthcare Center, Seoul, Republic of Korea
| | - You Jung Han
- Department of Obstetrics and Gynecology, Cheil General Hospital and Women's Healthcare Center, Dankook University College of Medicine, Seoul, Republic of Korea
| | - Min Hyoung Kim
- Department of Obstetrics and Gynecology, Cheil General Hospital and Women's Healthcare Center, Dankook University College of Medicine, Seoul, Republic of Korea
| | - Jin Hoon Chung
- Department of Obstetrics and Gynecology, Cheil General Hospital and Women's Healthcare Center, Dankook University College of Medicine, Seoul, Republic of Korea
| | - Jung Yeol Han
- Department of Obstetrics and Gynecology, Cheil General Hospital and Women's Healthcare Center, Dankook University College of Medicine, Seoul, Republic of Korea
| | - Moon Young Kim
- Department of Obstetrics and Gynecology, Cheil General Hospital and Women's Healthcare Center, Dankook University College of Medicine, Seoul, Republic of Korea
| | - Hyun Mee Ryu
- Laboratory of Medical Genetics, Medical Research Institute, Cheil General Hospital and Women's Healthcare Center, Seoul, Republic of Korea, .,Department of Obstetrics and Gynecology, Cheil General Hospital and Women's Healthcare Center, Dankook University College of Medicine, Seoul, Republic of Korea,
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25
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Keravnou A, Ioannides M, Loizides C, Tsangaras K, Achilleos A, Mina P, Kypri E, Hadjidaniel MD, Neofytou M, Kyriacou S, Sismani C, Koumbaris G, Patsalis PC. MeDIP combined with in-solution targeted enrichment followed by NGS: Inter-individual methylation variability of fetal-specific biomarkers and their implementation in a proof of concept study for NIPT. PLoS One 2018; 13:e0199010. [PMID: 29889893 PMCID: PMC5995407 DOI: 10.1371/journal.pone.0199010] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2018] [Accepted: 05/29/2018] [Indexed: 12/14/2022] Open
Abstract
DNA methylation is the most characterized epigenetic process exhibiting stochastic variation across different tissues and individuals. In non-invasive prenatal testing (NIPT) fetal specific methylated regions can potentially be used as biomarkers for the accurate detection of fetal aneuploidies. The aim of this study was the investigation of inter-individual methylation variability of previously reported fetal-specific markers and their implementation towards the development of a novel NIPT assay for the detection of trisomies 13, 18, and 21. Methylated DNA Immunoprecipitation (MeDIP) combined with in-solution targeted enrichment followed by NGS was performed in 29 CVS and 27 female plasma samples to assess inter-individual methylation variability of 331 fetal-specific differentially methylated regions (DMRs). The same approach was implemented for the NIPT of trisomies 13, 18 and 21 using spiked-in (n = 6) and pregnancy samples (n = 44), including one trisomy 13, one trisomy 18 and four trisomy 21. Despite the variability of DMRs, CVS samples showed statistically significant hypermethylation (p<2e-16) compared to plasma samples. Importantly, our assay correctly classified all euploid and aneuploid cases without any false positive results (n = 44). This work provides the starting point for the development of a NIPT assay based on a robust set of fetal specific biomarkers for the detection of fetal aneuploidies. Furthermore, the assay’s targeted nature significantly reduces the analysis cost per sample while providing high read depth at regions of interest increasing significantly its accuracy.
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Affiliation(s)
- Anna Keravnou
- Translational Genetics Team, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
| | | | | | | | | | | | | | - Michael D. Hadjidaniel
- Translational Genetics Team, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
| | - Maria Neofytou
- Translational Genetics Team, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
| | | | - Carolina Sismani
- Department of Cytogenetics and Genomics, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
| | | | - Philippos C. Patsalis
- Translational Genetics Team, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
- NIPD Genetics Ltd., Nicosia, Cyprus
- * E-mail:
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26
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Ulrich BC, Paweletz CP. Cell-Free DNA in Oncology: Gearing up for Clinic. Ann Lab Med 2018; 38:1-8. [PMID: 29071812 PMCID: PMC5700141 DOI: 10.3343/alm.2018.38.1.1] [Citation(s) in RCA: 39] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2017] [Revised: 08/09/2017] [Accepted: 09/21/2017] [Indexed: 01/06/2023] Open
Abstract
In the past several years, interest in the clinical utility of cell-free DNA as a noninvasive cancer biomarker has grown rapidly. Success in the development of plasma genotyping assays and other liquid biopsy assays has widened the scope of cell-free DNA use in research and the clinic. Already approved by the US Food and Drug Administration in the narrow context of epidermal growth factor receptor-mutated non-small cell lung cancer, plasma genotyping assays are currently being investigated in a wide array of clinical settings and modalities. These include plasma genotyping as a tool for early diagnosis, the detection of minimal residual disease, and the evaluation of treatment response/progression. In this review, we assess the clinical landscape of plasma genotyping assays and propose strategies for their further expansion into routine clinical care.
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Affiliation(s)
- Bryan C Ulrich
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.,Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, MA, USA
| | - Cloud P Paweletz
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.,Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, MA, USA.
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27
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Wilson SL, Robinson WP. Utility of DNA methylation to assess placental health. Placenta 2018; 64 Suppl 1:S23-S28. [DOI: 10.1016/j.placenta.2017.12.013] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/16/2017] [Revised: 12/12/2017] [Accepted: 12/13/2017] [Indexed: 12/18/2022]
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Lim JH, Lee BY, Kim JW, Han YJ, Chung JH, Kim MH, Kwak DW, Park SY, Choi HB, Ryu HM. Evaluation of extraction methods for methylated cell-free fetal DNA from maternal plasma. J Assist Reprod Genet 2018; 35:637-641. [PMID: 29423788 DOI: 10.1007/s10815-018-1114-8] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2017] [Accepted: 01/03/2018] [Indexed: 10/18/2022] Open
Abstract
PURPOSE Recently, fetal placenta-specific epigenetic regions (FSERs) have been identified for quantification of cell-free fetal DNA (cff-DNA) for non-invasive prenatal testing (NIPT). The aim of this study was to evaluate the efficiencies of a column-based kit and magnetic bead-based kit for quantification of methylated FSERs from maternal plasma. METHODS Maternal plasma was extracted from normal pregnant women within the gestational age of 10~13 weeks (n = 24). Total cell-free DNA (cf-DNA) was extracted using a column-based kit and magnetic bead-based kit from the plasma of the same pregnant woman, respectively. Methylated FSERs were enriched from the extracted total cf-DNA using a methyl-CpG-binding domain-based protein method. The four FSERs were simultaneously quantified by multiplex real-time polymerase chain reaction. RESULTS Methylated FSERs were detected in all samples extracted from both kits. However, the amplification of FSERs showed significant differences in the extraction efficiency of methylated FSERs between the two extraction methods. The Ct values of methylated FSERs extracted using the column-based kit were significantly lower than those obtained using the magnetic bead-based kit (P < 0.001 for all FSERs). The quantity of methylated FSERs was significantly higher for extracted DNA using the column-based kit than that extracted using the magnetic bead-based kit (P < 0.001 for all FSERs). Time and cost for the process of extraction were similar for the column kit and magnetic bead-based kit. CONCLUSIONS Our findings demonstrate that the column-based kit was more effective than the magnetic bead-based kit for isolation of methylated FSERs from maternal plasma as assessed by FSER detection.
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Affiliation(s)
- Ji Hyae Lim
- Laboratory of Medical Genetics, Medical Research Institute, Cheil General Hospital and Women's Healthcare Center, Seoul, South Korea
| | - Bom Yi Lee
- Laboratory of Medical Genetics, Medical Research Institute, Cheil General Hospital and Women's Healthcare Center, Seoul, South Korea
| | - Jin Woo Kim
- Laboratory of Medical Genetics, Medical Research Institute, Cheil General Hospital and Women's Healthcare Center, Seoul, South Korea
| | - You Jung Han
- Department of Obstetrics and Gynecology, Cheil General Hospital and Women's Healthcare Center, Dankook University College of Medicine, 1-19, Mookjung-dong, Chung-gu, Seoul, 100-380, South Korea
| | - Jin Hoon Chung
- Department of Obstetrics and Gynecology, Cheil General Hospital and Women's Healthcare Center, Dankook University College of Medicine, 1-19, Mookjung-dong, Chung-gu, Seoul, 100-380, South Korea
| | - Min Hyoung Kim
- Department of Obstetrics and Gynecology, Cheil General Hospital and Women's Healthcare Center, Dankook University College of Medicine, 1-19, Mookjung-dong, Chung-gu, Seoul, 100-380, South Korea
| | - Dong Wook Kwak
- Department of Obstetrics and Gynecology, Cheil General Hospital and Women's Healthcare Center, Dankook University College of Medicine, 1-19, Mookjung-dong, Chung-gu, Seoul, 100-380, South Korea
| | - So Yeon Park
- Laboratory of Medical Genetics, Medical Research Institute, Cheil General Hospital and Women's Healthcare Center, Seoul, South Korea
| | - Hee Back Choi
- Genes Laboratory, Molecular Diagnostic Institute, Gyeonggi-do, South Korea
| | - Hyun Mee Ryu
- Laboratory of Medical Genetics, Medical Research Institute, Cheil General Hospital and Women's Healthcare Center, Seoul, South Korea. .,Department of Obstetrics and Gynecology, Cheil General Hospital and Women's Healthcare Center, Dankook University College of Medicine, 1-19, Mookjung-dong, Chung-gu, Seoul, 100-380, South Korea.
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29
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Karami F, Noori-Daloii MR, Omidfar K, Tabrizi M, Hantooshzadeh S, Aleyasin A, Daneshpour M, Modarressi MH. Modified methylated DNA immunoprecipitation protocol for noninvasive prenatal diagnosis of Down syndrome. J Obstet Gynaecol Res 2018; 44:608-613. [PMID: 29377389 DOI: 10.1111/jog.13577] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2017] [Accepted: 12/06/2017] [Indexed: 11/30/2022]
Abstract
AIM Methylated DNA immunoprecipitation real-time quantitative polymerase chain reaction (MeDIP-real-time qPCR) has been introduced as noninvasive prenatal test that has shown absolute detection rate in the screening of Down syndrome. Herein, we aimed to propose a novel modification of MeDIP-qPCR and assess its potential to alleviate the overall cost of the test, being used in very early weeks of pregnancy, and develop it to a noninvasive prenatal diagnosis biosensor in future researches. METHODS Cell-free fetal DNA (cffDNA) isolated from 60 pregnant women, including 29 normal and 31 trisomy 21 pregnancies, were analyzed using proposed MeDIP protocol. Enriched methylated DNA sequences were amplified through real-time qPCR using eight fetal-specific primer pairs. The status of samples was determined through the calculation of D-value with the cutoff point of zero. RESULTS The sensitivity and specificity of the MeDIP protocols using nanoparticles were 100% and 100%, respectively. CONCLUSION Remarkable decrease in the price of MeDIP test per each patient would be a reasonable factor to confirm it on larger sample size. Moreover, the high detection rate of screening and the availability of the required instruments around the world make satisfactory reasons to be tested in earlier weeks of pregnancy, thanks to the high sensitivity of gold shell nanoparticles.
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Affiliation(s)
- Fatemeh Karami
- Department of Medical Genetics, Biophotonics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran
| | - Mohammad R Noori-Daloii
- Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Kobra Omidfar
- Biosensor Research Center, Endocrinology and Metabolite Molecular Cellular Science Institute, Tehran University of Medical Sciences, Tehran, Iran.,Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Biosensor Research Center, Endocrinology and Metabolite Molecular Cellular Science Institute, Tehran University of Medical Sciences, Tehran, Iran
| | - Mina Tabrizi
- Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Seddigheh Hantooshzadeh
- Faculty of Medicine, Vali-e-Asr Reproductive Health Research Center, Tehran University of Medical Sciences, Tehran, Iran
| | - Ashraf Aleyasin
- Department of Infertility, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran
| | - Maryam Daneshpour
- Biosensor Research Center, Endocrinology and Metabolite Molecular Cellular Science Institute, Tehran University of Medical Sciences, Tehran, Iran
| | - Mohammad H Modarressi
- Department of Medical Genetics, Biophotonics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran.,Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
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Wu DM, Ma LP, Song GL, Long Y, Liu HX, Liu Y, Ping J. Steroidogenic factor-1 hypermethylation in maternal rat blood could serve as a biomarker for intrauterine growth retardation. Oncotarget 2017; 8:96139-96153. [PMID: 29221193 PMCID: PMC5707087 DOI: 10.18632/oncotarget.21767] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2017] [Accepted: 09/21/2017] [Indexed: 01/10/2023] Open
Abstract
Intrauterine growth retardation (IUGR) is a common obstetric complication lacking an optimal method for prenatal screening. DNA methylation profile in maternal blood holds significant promise for prenatal screening. Here, we aimed to screen out potential IUGR biomarkers in maternal blood from the perspective of DNA methylation. The IUGR rat model was established by prenatal maternal undernutrition. High-throughput bisulfite sequencing of genomic DNA methylation followed by functional clustering analysis for differentially methylated region (DMR)-associated genes demonstrated that genes regulating transcription had the most significantly changed DNA methylation status in maternal blood with IUGR. Genes about apoptosis and placental development were also changed. Besides increased placental apoptosis, IUGR rats demonstrated the same hypermethylated CpG sites of steroidogenic factor-1 (SF-1, a DMR-associated transcription factor about placenta) promoter in maternal blood and placentae. Further, ff1b, the SF-1 ortholog, was knocked out in zebrafish by CRISPR/Cas9 technology. The knock-out zebrafish demonstrated developmental inhibition and increased IUGR rates, which confirmed the role of SF-1 in IUGR development. Finally, hypermethylated SF-1 was observed in human maternal blood of IUGR. This study firstly presented distinct DNA methylation profile in maternal blood of IUGR and showed hypermethylated SF-1 could be a potential IUGR biomarker in maternal rat blood.
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Affiliation(s)
- Dong-Mei Wu
- Department of Pharmacology, Wuhan University School of Basic Medical Sciences, Wuhan 430071, China
| | - Liang-Peng Ma
- Department of Pharmacology, Wuhan University School of Basic Medical Sciences, Wuhan 430071, China.,Department of Pharmacy, Wuhan First Hospital, Wuhan 430022, Hubei, China
| | - Gui-Li Song
- Key Laboratory of Biodiversity and Conservation of Aquatic Organism, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
| | - Yong Long
- Key Laboratory of Biodiversity and Conservation of Aquatic Organism, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
| | - Han-Xiao Liu
- Department of Pharmacology, Wuhan University School of Basic Medical Sciences, Wuhan 430071, China
| | - Yang Liu
- Department of Pharmacology, Wuhan University School of Basic Medical Sciences, Wuhan 430071, China
| | - Jie Ping
- Department of Pharmacology, Wuhan University School of Basic Medical Sciences, Wuhan 430071, China
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Intrauterine growth retardation-associated syncytin b hypermethylation in maternal rat blood revealed by DNA methylation array analysis. Pediatr Res 2017; 82:704-711. [PMID: 28604758 DOI: 10.1038/pr.2017.137] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/01/2016] [Accepted: 05/28/2017] [Indexed: 11/08/2022]
Abstract
BackgroundEmerging evidence suggests that DNA methylation in maternal blood is a promising target for intrauterine growth retardation (IUGR) screening, a common developmental toxicity. Here, we aimed to screen out IUGR-related DNA methylation status in maternal blood via high-throughput profiling.MethodsPregnant Wistar rats were subcutaneously administered nicotine (1 mg/kg) twice per day from gestational day (GD) 11 to GD20 to establish the IUGR model. MeDIP array assays and the following GO analysis were used to evaluate DNA methylation status in maternal blood. One placental development-associated gene was selected for further confirmation.ResultsGenes regulating the development of multiple organs and major body systems had changed DNA methylation frequencies in the maternal blood of IUGR rats. Placental development, which can affect the development of multiple fetal organs and induce IUGR, is a hypermethylated cluster consisting of four significantly changed genes, including syncytin b (Synb), Lrrc15, Met, and Tex19.1. With the most significant change, Synb hypermethylation in maternal blood was confirmed by bisulfite-sequencing PCR (BSP). Moreover, decreased Synb expression and histological changes were observed in IUGR placentae.ConclusionThe IUGR-associated DNA methylation profile in maternal blood, such as placenta-related Synb hypermethylation, provides evidence for further studies on possible IUGR biomarkers.
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Neocleous AC, Nicolaides KH, Schizas CN. Intelligent Noninvasive Diagnosis of Aneuploidy: Raw Values and Highly Imbalanced Dataset. IEEE J Biomed Health Inform 2017; 21:1271-1279. [PMID: 28026791 DOI: 10.1109/jbhi.2016.2608859] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The objective of this paper is to introduce a noninvasive diagnosis procedure for aneuploidy and to minimize the social and financial cost of prenatal diagnosis tests that are performed for fetal aneuploidies in an early stage of pregnancy. We propose a method by using artificial neural networks trained with data from singleton pregnancy cases, while undergoing first trimester screening. Three different datasets1 with a total of 122 362 euploid and 967 aneuploid cases were used in this study. The data for each case contained markers collected from the mother and the fetus. This study, unlike previous studies published by the authors for a similar problem differs in three basic principles: 1) the training of the artificial neural networks is done by using the markers' values in their raw form (unprocessed), 2) a balanced training dataset is created and used by selecting only a representative number of euploids for the training phase, and 3) emphasis is given to the financials and suggest hierarchy and necessity of the available tests. The proposed artificial neural networks models were optimized in the sense of reaching a minimum false positive rate and at the same time securing a 100% detection rate for Trisomy 21. These systems correctly identify other aneuploidies (Trisomies 13&18, Turner, and Triploid syndromes) at a detection rate greater than 80%. In conclusion, we demonstrate that artificial neural network systems can contribute in providing noninvasive, effective early screening for fetal aneuploidies with results that compare favorably to other existing methods.
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Vermeesch JR, Voet T, Devriendt K. Prenatal and pre-implantation genetic diagnosis. Nat Rev Genet 2017; 17:643-56. [PMID: 27629932 DOI: 10.1038/nrg.2016.97] [Citation(s) in RCA: 117] [Impact Index Per Article: 14.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The past decade has seen the development of technologies that have revolutionized prenatal genetic testing; that is, genetic testing from conception until birth. Genome-wide single-cell arrays and high-throughput sequencing analyses are dramatically increasing our ability to detect embryonic and fetal genetic lesions, and have substantially improved embryo selection for in vitro fertilization (IVF). Moreover, both invasive and non-invasive mutation scanning of the genome are helping to identify the genetic causes of prenatal developmental disorders. These advances are changing clinical practice and pose novel challenges for genetic counselling and prenatal care.
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Affiliation(s)
- Joris Robert Vermeesch
- Centre for Human Genetics, Department of Human Genetics, University of Leuven, 49 Herestraat, Leuven 3000, Belgium
| | - Thierry Voet
- Centre for Human Genetics, Department of Human Genetics, University of Leuven, 49 Herestraat, Leuven 3000, Belgium
| | - Koenraad Devriendt
- Centre for Human Genetics, Department of Human Genetics, University of Leuven, 49 Herestraat, Leuven 3000, Belgium
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34
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Neofytou MC, Tsangaras K, Kypri E, Loizides C, Ioannides M, Achilleos A, Mina P, Keravnou A, Sismani C, Koumbaris G, Patsalis PC. Targeted capture enrichment assay for non-invasive prenatal testing of large and small size sub-chromosomal deletions and duplications. PLoS One 2017; 12:e0171319. [PMID: 28158220 PMCID: PMC5291539 DOI: 10.1371/journal.pone.0171319] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2016] [Accepted: 01/18/2017] [Indexed: 02/06/2023] Open
Abstract
Noninvasive prenatal testing (NIPT) using whole genome and targeted sequencing has become increasingly accepted for clinical detection of Trisomy 21 and sex chromosome aneuploidies. Few studies have shown that sub-chromosomal deletions or duplications associated with genetic syndromes can also be detected in the fetus noninvasively. There are still limitations on these methodologies such as the detection of variants of unknown clinical significance, high number of false positives, and difficulties to detect small aberrations. We utilized a recently developed targeted sequencing approach for the development of a NIPT assay, for large and small size deletions/duplications, which overcomes these existing limitations. Artificial pregnancies with microdeletion/microduplication syndromes were created by spiking DNA from affected samples into cell free DNA (cfDNA) from non-pregnant samples. Unaffected spiked samples and normal pregnancies were used as controls. Target Capture Sequences (TACS) for seven syndromes were designed and utilized for targeted capture enrichment followed by sequencing. Data was analyzed using a statistical pipeline to identify deletions or duplications on targeted regions. Following the assay development a proof of concept study using 33 normal pregnancies, 21 artificial affected and 17 artificial unaffected pregnancies was carried out to test the sensitivity and specificity of the assay. All 21 abnormal spiked-in samples were correctly classified as subchromosomal aneuploidies while the 33 normal pregnancies or 17 normal spiked-in samples resulted in a false positive result. We have developed an NIPT assay for the detection of sub-chromosomal deletions and duplications using the targeted capture enrichment technology. This assay demonstrates high accuracy, high read depth of the genomic region of interest, and can identify deletions/duplications as small as 0.5 Mb. NIPT of fetal microdeletion/microduplication syndromes can be of enormous benefit in the management of pregnancies at risk both for prospective parents and health care providers.
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Affiliation(s)
- Maria C. Neofytou
- Translational Genetics Team, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
| | | | - Elena Kypri
- Translational Genetics Team, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
- NIPD Genetics Ltd, Nicosia, Cyprus
| | | | | | | | | | - Anna Keravnou
- Translational Genetics Team, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
| | - Carolina Sismani
- Department of Cytogenetics and Genomics, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
| | - George Koumbaris
- Translational Genetics Team, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
- NIPD Genetics Ltd, Nicosia, Cyprus
| | - Philippos C. Patsalis
- Translational Genetics Team, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
- NIPD Genetics Ltd, Nicosia, Cyprus
- * E-mail:
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Kazemi M, Salehi M, Kheirollahi M. MeDIP Real-Time qPCR has the Potential for Noninvasive Prenatal Screening of Fetal Trisomy 21. INTERNATIONAL JOURNAL OF MOLECULAR AND CELLULAR MEDICINE 2017; 6:13-21. [PMID: 28868265 PMCID: PMC5568188] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/31/2016] [Accepted: 02/01/2017] [Indexed: 11/25/2022]
Abstract
This study aimed to verify the reliability of the 7 tissue differentially methylated regions used in the methylated DNA immunoprecipitation (MeDIP) real- time quantitative polymerase chain reaction (real-time qPCR) based approach of fetal DNA in maternal blood to diagnosis of fetal trisomy 21. Forty pregnant women with high risk pregnancy who were referred after first or second trimester screening tests, were selected randomly. For each sample whole DNA extraction (mother and fetus), fragmentation of DNA, immunoprecipitation of methylated DNA and real- time qPCR using 7 primer pairs was performed. D-value for each sample was calculated using the following formula D = -4.908+ 0.254 XEP1+ 0.409 XEP4+ 0.793 XEP5+ 0.324 XEP6+ 0.505 XEP7+ 0.508 XEP9+ 0.691 XEP12. In all normal cases, D value was negative, while it was positive in all trisomy cases. Therefore, all normal and trisomy 21 cases were classified correctly which correspond to 100% specificity and 100% sensitivity for this method. The MeDIP real-time qPCR method has provided the opportunity for noninvasive prenatal diagnosis of fetal trisomy 21 to be potentially employed into the routine practice of diagnostic laboratories.
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Affiliation(s)
- Mohammad Kazemi
- Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.,Medical Genetic Center of Genome, Isfahan, Iran.,Pediatric Inherited Diseases Research Center, Research Institute for Primordial Prevention of Noncommuni-cable Disease, Isfahan University of Medical Sciences, Isfahan, Iran.
| | - Mansoor Salehi
- Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.,Medical Genetic Center of Genome, Isfahan, Iran.,Pediatric Inherited Diseases Research Center, Research Institute for Primordial Prevention of Noncommuni-cable Disease, Isfahan University of Medical Sciences, Isfahan, Iran.,Corresponding author: Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran. E. mail: .
| | - Majid Kheirollahi
- Pediatric Inherited Diseases Research Center, Research Institute for Primordial Prevention of Noncommuni-cable Disease, Isfahan University of Medical Sciences, Isfahan, Iran.
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Whole-genome fetal and maternal DNA methylation analysis using MeDIP-NGS for the identification of differentially methylated regions. Genet Res (Camb) 2016; 98:e15. [PMID: 27834155 PMCID: PMC6865150 DOI: 10.1017/s0016672316000136] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
DNA methylation is an epigenetic marker that has been shown to vary significantly across different tissues. Taking advantage of the methylation differences between placenta-derived cell-free DNA and maternal blood, several groups employed different approaches for the discovery of fetal-specific biomarkers. The aim of this study was to analyse whole-genome fetal and maternal methylomes in order to identify and confirm the presence of differentially methylated regions (DMRs). We have initially utilized methylated DNA immunoprecipitation (MeDIP) and next-generation sequencing (NGS) to identify genome-wide DMRs between chorionic villus sampling (CVS) and female non-pregnant plasma (PL) and peripheral blood (WBF) samples. Next, using specific criteria, 331 fetal-specific DMRs were selected and confirmed in eight CVS, eight WBF and eight PL samples by combining MeDIP and in-solution targeted enrichment followed by NGS. Results showed higher enrichment in CVS samples as compared to both WBF and PL samples, confirming the distinct methylation levels between fetal and maternal DNA for the selected DMRs. We have successfully implemented a novel approach for the discovery and confirmation of a significant number of fetal-specific DMRs by combining for the first time MeDIP and in-solution targeted enrichment followed by NGS. The implementation of this double-enrichment approach is highly efficient and enables the detailed analysis of multiple DMRs by targeted NGS. Also, this is, to our knowledge, the first reported application of MeDIP on plasma samples, which leverages the implementation of our enrichment methodology in the detection of fetal abnormalities in maternal plasma.
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37
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The dynamic changes of DNA methylation in primordial germ cell differentiation. Gene 2016; 591:305-12. [DOI: 10.1016/j.gene.2016.06.036] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2016] [Revised: 06/07/2016] [Accepted: 06/15/2016] [Indexed: 01/19/2023]
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38
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Use of the STR loci D18S53, D18S59, and D18S488 in the diagnosis of Edwards’ syndrome. Genes Genomics 2016. [DOI: 10.1007/s13258-016-0412-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
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Bock C, Halbritter F, Carmona FJ, Tierling S, Datlinger P, Assenov Y, Berdasco M, Bergmann AK, Booher K, Busato F, Campan M, Dahl C, Dahmcke CM, Diep D, Fernández AF, Gerhauser C, Haake A, Heilmann K, Holcomb T, Hussmann D, Ito M, Kläver R, Kreutz M, Kulis M, Lopez V, Nair SS, Paul DS, Plongthongkum N, Qu W, Queirós AC, Reinicke F, Sauter G, Schlomm T, Statham A, Stirzaker C, Strogantsev R, Urdinguio RG, Walter K, Weichenhan D, Weisenberger DJ, Beck S, Clark SJ, Esteller M, Ferguson-Smith AC, Fraga MF, Guldberg P, Hansen LL, Laird PW, Martín-Subero JI, Nygren AOH, Peist R, Plass C, Shames DS, Siebert R, Sun X, Tost J, Walter J, Zhang K. Quantitative comparison of DNA methylation assays for biomarker development and clinical applications. Nat Biotechnol 2016; 34:726-37. [PMID: 27347756 DOI: 10.1038/nbt.3605] [Citation(s) in RCA: 228] [Impact Index Per Article: 25.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2015] [Accepted: 05/10/2016] [Indexed: 02/08/2023]
Abstract
DNA methylation patterns are altered in numerous diseases and often correlate with clinically relevant information such as disease subtypes, prognosis and drug response. With suitable assays and after validation in large cohorts, such associations can be exploited for clinical diagnostics and personalized treatment decisions. Here we describe the results of a community-wide benchmarking study comparing the performance of all widely used methods for DNA methylation analysis that are compatible with routine clinical use. We shipped 32 reference samples to 18 laboratories in seven different countries. Researchers in those laboratories collectively contributed 21 locus-specific assays for an average of 27 predefined genomic regions, as well as six global assays. We evaluated assay sensitivity on low-input samples and assessed the assays' ability to discriminate between cell types. Good agreement was observed across all tested methods, with amplicon bisulfite sequencing and bisulfite pyrosequencing showing the best all-round performance. Our technology comparison can inform the selection, optimization and use of DNA methylation assays in large-scale validation studies, biomarker development and clinical diagnostics.
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40
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Koumbaris G, Kypri E, Tsangaras K, Achilleos A, Mina P, Neofytou M, Velissariou V, Christopoulou G, Kallikas I, González-Liñán A, Benusiene E, Latos-Bielenska A, Marek P, Santana A, Nagy N, Széll M, Laudanski P, Papageorgiou EA, Ioannides M, Patsalis PC. Cell-Free DNA Analysis of Targeted Genomic Regions in Maternal Plasma for Non-Invasive Prenatal Testing of Trisomy 21, Trisomy 18, Trisomy 13, and Fetal Sex. Clin Chem 2016; 62:848-55. [DOI: 10.1373/clinchem.2015.252502] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2015] [Accepted: 03/21/2016] [Indexed: 12/19/2022]
Abstract
Abstract
BACKGROUND
There is great need for the development of highly accurate cost effective technologies that could facilitate the widespread adoption of noninvasive prenatal testing (NIPT).
METHODS
We developed an assay based on the targeted analysis of cell-free DNA for the detection of fetal aneuploidies of chromosomes 21, 18, and 13. This method enabled the capture and analysis of selected genomic regions of interest. An advanced fetal fraction estimation and aneuploidy determination algorithm was also developed. This assay allowed for accurate counting and assessment of chromosomal regions of interest. The analytical performance of the assay was evaluated in a blind study of 631 samples derived from pregnancies of at least 10 weeks of gestation that had also undergone invasive testing.
RESULTS
Our blind study exhibited 100% diagnostic sensitivity and specificity and correctly classified 52/52 (95% CI, 93.2%–100%) cases of trisomy 21, 16/16 (95% CI, 79.4%–100%) cases of trisomy 18, 5/5 (95% CI, 47.8%–100%) cases of trisomy 13, and 538/538 (95% CI, 99.3%–100%) normal cases. The test also correctly identified fetal sex in all cases (95% CI, 99.4%–100%). One sample failed prespecified assay quality control criteria, and 19 samples were nonreportable because of low fetal fraction.
CONCLUSIONS
The extent to which free fetal DNA testing can be applied as a universal screening tool for trisomy 21, 18, and 13 depends mainly on assay accuracy and cost. Cell-free DNA analysis of targeted genomic regions in maternal plasma enables accurate and cost-effective noninvasive fetal aneuploidy detection, which is critical for widespread adoption of NIPT.
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Affiliation(s)
| | | | | | | | | | | | | | | | | | | | - Egle Benusiene
- Department of Human and Medical Genetics, Faculty of Medicine, Vilnius University, Vilnius, Lithuania
| | | | - Pietryga Marek
- Gynecology and Obstetrics, Poznan University of Medical Sciences, Poland
| | - Alfredo Santana
- Clinical Genetics, Childhood Hospital Materno-Infantil, Las Palmas GC, Canary Islands, Spain
| | - Nikoletta Nagy
- Department of Medical Genetics, University of Szeged, Szeged, Hungary
| | - Márta Széll
- Department of Medical Genetics, University of Szeged, Szeged, Hungary
| | - Piotr Laudanski
- Department of Perinatology and Obstetrics Medical University of Bialystok, Poland
| | | | | | - Philippos C Patsalis
- NIPD Genetics, Ltd, Nicosia, Cyprus
- The Cyprus Institute of Neurology and Genetics
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41
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Wei Z, Shah N, Deng C, Xiao X, Zhong T, Li X. Circulating DNA addresses cancer monitoring in non small cell lung cancer patients for detection and capturing the dynamic changes of the disease. SPRINGERPLUS 2016; 5:531. [PMID: 27186495 PMCID: PMC4844578 DOI: 10.1186/s40064-016-2141-5] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/13/2016] [Accepted: 04/12/2016] [Indexed: 11/18/2022]
Abstract
Purpose Monitoring of key markers for lung cancer detection and tracking of acquired drug resistance is critical for the management of the disease. We aim to ascertain the value of monitoring both total cell free DNA concentrations and mutant EGFR DNA content within diverse groups of individuals most vulnerable to the disease. Methods We proposed longitudinal monitoring of circulating DNA using digital PCR. Circulating DNA present in peripheral blood can be obtained non-invasively and provide timely disease status update. 25 heavy smokers and 50 patients undergoing TKI therapy were recruited. Peripheral blood specimens were taken at different time points and their circulating DNA were analyzed and quantified. Results Significant higher concentrations of total cell free DNA were detected when compared with healthy high-risk individuals. Levels were stable throughout the treatment cycles, which makes it potentially a useful tool for patient stratification. Concurrent mutant T790M DNA detection of lung cancer patients at baseline achieved 82 % concordance with matched tissue analysis. Samples initially negative for the T790M gene mutation that became positive during treatment were corroborated with a repeat biopsy. The results showed its usefulness for serial monitoring. Conclusion Monitoring of circulating DNA addresses the need for disease detection and shows the ability to capture the dynamic changes of the disease. Electronic supplementary material The online version of this article (doi:10.1186/s40064-016-2141-5) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Zhangjing Wei
- Department of Diagnostic Medicine, JingMen NO.1 People's Hospital, JingMen, People's Republic of China
| | - Nirej Shah
- Department of Clinical Medicine, JingChu University of Technology, JingMen, People's Republic of China
| | - Chong Deng
- Department of Diagnostic Medicine, JingMen NO.1 People's Hospital, JingMen, People's Republic of China
| | - Xuehui Xiao
- Department of Diagnostic Medicine, JingMen NO.1 People's Hospital, JingMen, People's Republic of China
| | - Tenglang Zhong
- Department of Diagnostic Medicine, JingMen NO.1 People's Hospital, JingMen, People's Republic of China
| | - Xiansong Li
- Department of Neurosurgery, JingZhou Hospital, Tongji Medical College, Huazhong University of Science and Technology (HUST), Renmin Road 1, JingZhou, 434020 People's Republic of China
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Abstract
Advances in next-generation sequencing have provided new insights and new therapeutic options for patients with cancer. However, assessment of genomic aberrations, which is required for precision medicine, has been challenging because of the difficulties in capturing intratumoral heterogeneity and in real-time assessment of tumors. Recent advances in technology have enabled detection and analysis of cell-free DNA in cancer patients, which provides real-time assessment of tumor evolution. Here, we review the recent advances in our understanding of the clinical utility of cell-free DNA and the future directions for its use in cancer management.
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Affiliation(s)
- Shumei Kato
- Department of Investigational Cancer Therapeutics, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, FC8.2018, Box 0455, Houston, TX 77030, USA
| | - Filip Janku
- Department of Investigational Cancer Therapeutics, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, FC8.2018, Box 0455, Houston, TX 77030, USA
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43
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Zhang J, Zhang B. Second-generation non-invasive high-throughput DNA sequencing technology in the screening of Down's syndrome in advanced maternal age women. Biomed Rep 2016; 4:715-718. [PMID: 27313855 DOI: 10.3892/br.2016.653] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2015] [Accepted: 01/27/2016] [Indexed: 01/29/2023] Open
Abstract
The aim of the present study was to evaluate the efficacy of using non-invasive DNA testing technology in screening Down's syndrome among women of advanced maternal age (AMA) and to provide evidence for prenatal screening of Down's syndrome. With a double-blind design, 8 ml of peripheral venous blood samples were collected from 87 women aged ≥35 years after 12 weeks of pregnancy. All cases were recorded with unique identification cards with clinical details and followed up until delivery. All the non-invasive prenatal testing results were confirmed by amniotic fluid fetal karyotyping (the gold standard of aneuploidy test), follow-up examination by neonatologists or neonatal blood karyotyping. The sensitivity, specificity and other indicators of non-invasive DNA testing technology were calculated based on the data of 87 women of AMA. Among the 87 women of AMA, 5 were cases with abnormal numbers of chromosomes (3 cases of trisomy 21, 1 case of trisomy 18 and 1 case of 47, XXX). The sensitivity and specificity reached 100% for trisomy 21, trisomy 18 and 47, XXX. The present study supports that non-invasive DNA testing is a useful method of AMA screening of Down's syndrome with 100% accuracy. Therefore, it can be used as an important alternative screening method for Down's syndrome in women of AMA.
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Affiliation(s)
- Jiao Zhang
- Obstetrics Department, Obstetrics and Gynecology Hospital of Fudan University, Shanghai 200011, P.R. China
| | - Bin Zhang
- Obstetrics Department, Obstetrics and Gynecology Hospital of Fudan University, Shanghai 200011, P.R. China
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44
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Abstract
Cell-free fetal DNA screening for Down syndrome has gained rapid acceptance over the past few years with increasing market penetration. Three main laboratory methodologies are currently used: a massive parallel shotgun sequencing (MPSS), a targeted massive parallel sequencing (t-MPS) and a single nucleotide polymorphism (SNP) based approach. Although each of these technologies has its own advantages and disadvantages, the performance of all was shown to be comparable and superior to that of traditional first-trimester screening for the detection of trisomy 21 in a routine prenatal population. Differences in performance were predominantly shown for chromosomal anomalies other than trisomy 21. Understanding the limitations and benefits of each technology is essential for proper counseling to patients. These technologies, as well as few investigational technologies described in this review, carry a great potential beyond screening for the common aneuploidies.
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Affiliation(s)
- Pe'er Dar
- Department of Obstetrics & Gynecology and Women's Health, Montefiore Medical Center, Albert Einstein College of Medicine, 1695 Eastchester Road, Bronx, New York 10461, USA
| | - Hagit Shani
- Department of Obstetrics & Gynecology and Women's Health, Montefiore Medical Center, Albert Einstein College of Medicine, 1695 Eastchester Road, Bronx, New York 10461, USA
| | - Mark I Evans
- Department of Obstetrics and Gynecology, Mt. Sinai School of Medicine, New York, NY, USA; Comprehensive Genetics and Fetal Medicine Foundation of America, 131 East 65th Street, New York, NY 10065, USA.
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45
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Lee DE, Lim JH, Kim MH, Park SY, Ryu HM. Novel Epigenetic Markers on Chromosome 21 for Noninvasive Prenatal Testing of Fetal Trisomy 21. J Mol Diagn 2016; 18:378-387. [PMID: 26947512 DOI: 10.1016/j.jmoldx.2015.12.002] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2015] [Revised: 11/25/2015] [Accepted: 12/09/2015] [Indexed: 10/22/2022] Open
Abstract
Until now, fetal placenta-specific epigenetic markers for noninvasive prenatal testing of fetal trisomy 21 (T21) have been identified based only on differences in tissue-specific epigenetic characteristics between placenta and maternal blood, but these characteristics have not been validated in T21 placenta. We aimed to discover novel epigenetic markers on chromosome 21 that show a hypermethylated pattern in fetal placenta compared with blood, regardless of the presence of T21. We performed a high-resolution tiling array analysis of chromosome 21 using the methylated-CpG binding domain protein-based method. We identified 93 epigenetic regions that showed fetal placenta-specific differential methylation patterns; among these, three regions showed fetal placenta-specific methylation patterns in T21 placenta samples. The methylation patterns of these three regions in the array were confirmed by bisulfite direct sequencing. The three regions were detectable in first-trimester maternal plasma. Moreover, a combination of their methylation ratio achieved high diagnostic accuracy for noninvasive prenatal testing of fetal T21 by further statistical analysis. These three novel regions with fetal placenta-specific differential methylation patterns on chromosome 21 were identified irrespective of the presence of T21. Our findings suggest that epigenetic characteristics of markers according to the presence or absence of T21 should be considered in the development of noninvasive prenatal testing of fetal T21 using fetal placenta-specific epigenetic markers.
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Affiliation(s)
- Da Eun Lee
- Laboratory of Medical Genetics, Medical Research Institute, Cheil General Hospital and Women's Healthcare Center, Seoul, Republic of Korea
| | - Ji Hyae Lim
- Laboratory of Medical Genetics, Medical Research Institute, Cheil General Hospital and Women's Healthcare Center, Seoul, Republic of Korea
| | - Min Hyoung Kim
- Department of Obstetrics and Gynecology, Cheil General Hospital and Women's Healthcare Center, Dankook University College of Medicine, Seoul, Republic of Korea
| | - So Yeon Park
- Laboratory of Medical Genetics, Medical Research Institute, Cheil General Hospital and Women's Healthcare Center, Seoul, Republic of Korea.
| | - Hyun Mee Ryu
- Laboratory of Medical Genetics, Medical Research Institute, Cheil General Hospital and Women's Healthcare Center, Seoul, Republic of Korea; Department of Obstetrics and Gynecology, Cheil General Hospital and Women's Healthcare Center, Dankook University College of Medicine, Seoul, Republic of Korea.
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46
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Affiliation(s)
- Felix C.K. Wong
- Centre for Research into Circulating Fetal Nucleic Acids, Li Ka Shing Institute of Health Sciences, and
- Department of Chemical Pathology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China; ,
| | - Y.M. Dennis Lo
- Centre for Research into Circulating Fetal Nucleic Acids, Li Ka Shing Institute of Health Sciences, and
- Department of Chemical Pathology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China; ,
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47
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Noninvasive fetal trisomy detection by multiplexed semiconductor sequencing: a barcoding analysis strategy. J Hum Genet 2015; 61:247-52. [PMID: 26657936 DOI: 10.1038/jhg.2015.142] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2015] [Revised: 10/27/2015] [Accepted: 10/29/2015] [Indexed: 01/09/2023]
Abstract
Noninvasive prenatal detection of fetal chromosomal aneuploidies by high-throughput next-generation sequencing proves to be accurate and sensitive. Currently, most of the data analysis methods involve a Z-score test based on the reference distribution of at least dozens of normal samples. This is not only costly but also time consuming. Moreover, as the experimental condition varies between every single run, noises cannot be eliminated and will skew the results. In order to overcome these drawbacks, we have proposed a new analytical strategy based on the multiplex barcoding sequencing of both normal and unknown samples in a single run on Ion Torrent PGM. In this method, only one normal sample is required. By applying this method to 13 single runs with a total number of 44 samples, we achieved the sensitivity and specificity of 100 and 95.181% for T13 detection, 100 and 100% for T18 detection, 90 and 100% for T21 detection, respectively.
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48
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Ocaña A, Díez-González L, García-Olmo DC, Templeton AJ, Vera-Badillo F, José Escribano M, Serrano-Heras G, Corrales-Sánchez V, Seruga B, Andrés-Pretel F, Pandiella A, Amir E. Circulating DNA and Survival in Solid Tumors. Cancer Epidemiol Biomarkers Prev 2015; 25:399-406. [PMID: 26604269 DOI: 10.1158/1055-9965.epi-15-0893] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2015] [Accepted: 11/04/2015] [Indexed: 11/16/2022] Open
Abstract
BACKGROUND The ability to undertake molecular analysis to inform on prognosis and predictors of response to therapy is limited by accessibility of tissue. Measurement of total circulating free DNA (cfDNA) or circulating tumor DNA (ctDNA) in peripheral blood may allow easier access to tumor material and help to predict clinical outcomes. METHODS A systematic review of electronic databases identified publications exploring the association between cfDNA or ctDNA and overall survival (OS) in solid tumors. HRs for OS were extracted from multivariable analyses and included in a meta-analysis. Pooled HRs were computed and weighted using generic inverse variance and random-effect modeling. For studies not reporting multivariable analyses, univariable ORs were estimated from Kaplan-Meier curves for OS at 1 and 3 years. RESULTS Thirty-nine studies comprising 4,052 patients were included in the analysis. Detection of ctDNA was associated with a significantly worse OS in multivariable analyses [HR, 2.70; 95% confidence interval (CI), 2.02-3.61; P < 0.001). Similar results were observed in the univariable analyses at 3 and 1 year (OR, 4.83; 95% CI, 3.20-7.28; P < 0.001).There was also a statistically significant association between high total cfDNA and worse OS for studies reporting multivariable and univariate data at 3 years (HR, 1.91; 95% CI, 1.59-2.29; P < 0.001 and OR, 2.82; 95% CI, 1.93-4.13; P < 0.001, respectively). CONCLUSIONS High levels of total cfDNA and presence of ctDNA are associated with worse survival in solid tumors. IMPACT Circulating DNA is associated with worse outcome in solid tumors.
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Affiliation(s)
- Alberto Ocaña
- Translational Oncology Unit, Albacete University Hospital, Albacete, Spain.
| | | | | | - Arnoud J Templeton
- Department of Medical Oncology and Hematology, Kantonsspital St. Gallen, St. Gallen, Switzerland
| | - Francisco Vera-Badillo
- Divisions of Medical Oncology and Hematology, Princess Margaret Cancer Centre, Department of Medicine, University of Toronto, Toronto, Canada
| | | | | | | | - Bostjan Seruga
- Department of Medical Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia
| | | | | | - Eitan Amir
- Divisions of Medical Oncology and Hematology, Princess Margaret Cancer Centre, Department of Medicine, University of Toronto, Toronto, Canada
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Kotsopoulou I, Tsoplou P, Mavrommatis K, Kroupis C. Non-invasive prenatal testing (NIPT): limitations on the way to become diagnosis. ACTA ACUST UNITED AC 2015. [PMID: 29540035 DOI: 10.1515/dx-2015-0002] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
With the discovery of existing circulating cell-free fetal DNA (ccffDNA) in maternal plasma and the advent of next-generation sequencing (NGS) technology, there is substantial hope that prenatal diagnosis will become a predominately non-invasive process in the future. At the moment, non-invasive prenatal testing (NIPT) is available for high-risk pregnancies with significant better sensitivity and specificity than the other existing non-invasive methods (biochemical and ultrasonographical). Mainly it is performed by NGS methods in a few commercial labs worldwide. However, it is expected that many other labs will offer analogous services in the future in this fast-growing field with a multiplicity of in-house methods (e.g., epigenetic, etc.). Due to various limitations of the available methods and technologies that are explained in detail in this manuscript, NIPT has not become diagnostic yet and women may still need to undergo risky invasive procedures to verify a positive finding or to secure (or even expand) a negative one. Efforts have already started to make the NIPT technologies more accurate (even at the level of a complete fetal genome) and cheaper and thus more affordable, in order to become diagnostic screening tests for all pregnancies in the near future.
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Affiliation(s)
- Ioanna Kotsopoulou
- 1Department of Clinical Biochemistry, Attikon University General Hospital, University of Athens Medical School, Athens, Greece
| | | | | | - Christos Kroupis
- 1Department of Clinical Biochemistry, Attikon University General Hospital, University of Athens Medical School, Athens, Greece
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50
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Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing. PLoS One 2015; 10:e0135058. [PMID: 26247357 PMCID: PMC4527772 DOI: 10.1371/journal.pone.0135058] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2015] [Accepted: 07/16/2015] [Indexed: 02/08/2023] Open
Abstract
INTRODUCTION Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity. METHODS Performance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood. RESULTS The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood. CONCLUSION Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed. Finally, we showed that bisulfite amplicon sequencing is a suitable approach for methylation analysis of targeted regions.
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