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Thomas Mock - One of the best experts on this subject based on the ideXlab platform.

  • integrative analysis of chloroplast DNA methylation in a marine alga saccharina japonica
    Plant Molecular Biology, 2021
    Co-Authors: Linhong Teng, Wentao Han, Xiao Fan, Xiaowen Zhang, Yitao Wang, Sadequr Rahman, Matteo Pellegrini, Thomas Mock
    Abstract:

    KEY MESSAGE We applied an integrative approach using multiple methods to verify cytosine methylation in the chloroplast DNA of the multicellular brown alga Saccharina japonica. Cytosine DNA methylation is a heritable process which plays important roles in regulating development throughout the life cycle of an organism. Although methylation of nuclear DNA has been studied extensively, little is known about the state and role of DNA methylation in chloroplast genomes, especially in marine algae. Here, we have applied an integrated approach encompassing whole-genome bisulfite sequencing, Methylated DNA Immunoprecipitation, gene co-expression networks and photophysiological analyses to provide evidence for the role of chloroplast DNA methylation in a marine alga, the multicellular brown alga Saccharina japonica. Although the overall methylation level was relatively low in the chloroplast genome of S. japonica, gametophytes exhibited higher methylation levels than sporophytes. Gene-specific bisulfite-cloning sequencing provided additional evidence for the methylation of key photosynthetic genes. Many of them were highly expressed in sporophytes whereas genes involved in transcription, translation and biosynthesis were strongly expressed in gametophytes. Nucleus-encoded photosynthesis genes were co-expressed with their chloroplast-encoded counterparts potentially contributing to the higher photosynthetic performance in sporophytes compared to gametophytes where these co-expression networks were less pronounced. A nucleus-encoded DNA methyltransferase of the DNMT2 family is assumed to be responsible for the methylation of the chloroplast genome because it is predicted to possess a plastid transit peptide.

  • integrative analysis of chloroplast DNA methylation in a marine alga saccharina japonica
    Plant Molecular Biology, 2021
    Co-Authors: Linhong Teng, Wentao Han, Xiao Fan, Xiaowen Zhang, Yitao Wang, Sadequr Rahman, Matteo Pellegrini, Thomas Mock
    Abstract:

    We applied an integrative approach using multiple methods to verify cytosine methylation in the chloroplast DNA of the multicellular brown alga Saccharina japonica. Cytosine DNA methylation is a heritable process which plays important roles in regulating development throughout the life cycle of an organism. Although methylation of nuclear DNA has been studied extensively, little is known about the state and role of DNA methylation in chloroplast genomes, especially in marine algae. Here, we have applied an integrated approach encompassing whole-genome bisulfite sequencing, Methylated DNA Immunoprecipitation, gene co-expression networks and photophysiological analyses to provide evidence for the role of chloroplast DNA methylation in a marine alga, the multicellular brown alga Saccharina japonica. Although the overall methylation level was relatively low in the chloroplast genome of S. japonica, gametophytes exhibited higher methylation levels than sporophytes. Gene-specific bisulfite-cloning sequencing provided additional evidence for the methylation of key photosynthetic genes. Many of them were highly expressed in sporophytes whereas genes involved in transcription, translation and biosynthesis were strongly expressed in gametophytes. Nucleus-encoded photosynthesis genes were co-expressed with their chloroplast-encoded counterparts potentially contributing to the higher photosynthetic performance in sporophytes compared to gametophytes where these co-expression networks were less pronounced. A nucleus-encoded DNA methyltransferase of the DNMT2 family is assumed to be responsible for the methylation of the chloroplast genome because it is predicted to possess a plastid transit peptide.

Evdokia Tsaliki - One of the best experts on this subject based on the ideXlab platform.

  • variability of ffDNA in maternal plasma does not prevent correct classification of trisomy 21 using medip qpcr methodology
    Prenatal Diagnosis, 2013
    Co-Authors: Skevi Kyriakou, Elena Kypri, Christiana Spyrou, Evdokia Tsaliki, Voula Velissariou, Elisavet A Papageorgiou, Philippos C Patsalis
    Abstract:

    Objective The goal of this study is to evaluate the amount of free fetal DNA (ffDNA), total DNA, and ‘fetal fraction’ found in maternal plasma and whether these influence the enrichment ratios of differentially Methylated regions (DMRs) and the correct classification of trisomy 21 using the Methylated DNA Immunoprecipitation-quantitative polymerase chain reaction (MeDIP-qPCR)-based noninvasive prenatal diagnostic methodology applied in peripheral blood. Methods Absolute quantification of ffDNA using DYS14 and total DNA using β-globin was applied in 83 maternal plasma samples. The quantification values for all 83 samples were correlated with the enrichment ratios of all seven DMRs and D-values that were obtained from the diagnostic formula of MeDIP-qPCR method. Results Our analysis concluded that trisomy 21 samples had significantly higher ffDNA and total DNA levels compared with those of normal samples. Enrichment ratios of the majority of DMRs studied exhibited no association with ffDNA, total DNA, and ‘fetal fraction’, and only a small portion of DMRs exhibited moderate association. Correlation studies of ffDNA, total DNA, and fetal fraction with the diagnostic D-value showed weak to no association but without affecting the classification of trisomy 21. Conclusion Overall, the variability of ffDNA and total DNA among maternal samples does not affect the correct trisomy 21 classification using MeDIP-qPCR methodology applied in peripheral blood. © 2013 John Wiley & Sons, Ltd.

  • medip real time qpcr of maternal peripheral blood reliably identifies trisomy 21
    Prenatal Diagnosis, 2012
    Co-Authors: Skevi Kyriakou, Elena Kypri, Christiana Spyrou, Evdokia Tsaliki, Elisavet A Papageorgiou, George Koumbaris, Chrysovalanto Sotiriou, Evi Touvana, Anna Keravnou
    Abstract:

    Objective To reevaluate the efficiency of the 12 differentially Methylated regions (DMRs) used in the Methylated DNA Immunoprecipitation (MeDIP) real-time quantitative polymerase chain reaction (real-time qPCR) based approach, develop an improved version of the diagnostic formula and perform a larger validation study. Methods Twelve selected DMRs were checked for copy number variants in the Database of Genomic Variants. The DMRs located within copy number variants were excluded from the analysis. One hundred and seventy-five maternal peripheral blood samples were used to reconstruct and evaluate the new diagnostic formula and for a larger-scale blinded validation study using MeDIP real-time qPCR. Results Seven DMRs entered the final model of the prediction equation and a larger blinded validation study demonstrated 100% sensitivity and 99.2% specificity. No significant evidence for association was observed between cell free fetal DNA concentration and D value. Conclusion The MeDIP real-time qPCR method for noninvasive prenatal diagnosis of trisomy 21 was confirmed and revalidated in 175 samples with satisfactory results demonstrating that it is accurate and reproducible. We are currently working towards simplification of the method to make it more robust and therefore easily, accurately, and rapidly reproduced and adopted by other laboratories. Nevertheless, larger scale validation studies are necessary before the MeDIP real-time qPCR-based method could be applied in clinical practice. © 2012 John Wiley & Sons, Ltd.

  • fetal specific DNA methylation ratio permits noninvasive prenatal diagnosis of trisomy 21
    Nature Medicine, 2011
    Co-Authors: Elisavet A Papageorgiou, Evdokia Tsaliki, Voula Velissariou, Alex Karagrigoriou, Nigel P Carter, Philippos C Patsalis
    Abstract:

    Noninvasive testing for Down's syndrome (trisomy 21) would greatly reduce the risks associated with the more invasive techniques used currently. Earlier identification of differentially Methylated regions between fetal DNA and maternal peripheral blood has now enabled Elisavet Papageorgiou and her colleagues to develop a strategy involving Methylated DNA Immunoprecipitation in combination with real-time quantitative PCR that discriminates normal from trisomy 21 cases in maternal peripheral blood with high sensitivity.

  • fetal specific DNA methylation ratio permits noninvasive prenatal diagnosis of trisomy 21
    Nature Medicine, 2011
    Co-Authors: Elisavet A Papageorgiou, Evdokia Tsaliki, Voula Velissariou, Alex Karagrigoriou, Nigel P Carter, Philippos C Patsalis
    Abstract:

    The trials performed worldwide toward noninvasive prenatal diagnosis (NIPD) of Down's syndrome (or trisomy 21) have shown the commercial and medical potential of NIPD compared to the currently used invasive prenatal diagnostic procedures. Extensive investigation of methylation differences between the mother and the fetus has led to the identification of differentially Methylated regions (DMRs). In this study, we present a strategy using the Methylated DNA Immunoprecipitation (MeDiP) methodology in combination with real-time quantitative PCR (qPCR) to achieve fetal chromosome dosage assessment, which can be performed noninvasively through the analysis of fetal-specific DMRs. We achieved noninvasive prenatal detection of trisomy 21 by determining the methylation ratio of normal and trisomy 21 cases for each tested fetal-specific DMR present in maternal peripheral blood, followed by further statistical analysis. The application of this fetal-specific methylation ratio approach provided correct diagnosis of 14 trisomy 21 and 26 normal cases.

Linhong Teng - One of the best experts on this subject based on the ideXlab platform.

  • integrative analysis of chloroplast DNA methylation in a marine alga saccharina japonica
    Plant Molecular Biology, 2021
    Co-Authors: Linhong Teng, Wentao Han, Xiao Fan, Xiaowen Zhang, Yitao Wang, Sadequr Rahman, Matteo Pellegrini, Thomas Mock
    Abstract:

    KEY MESSAGE We applied an integrative approach using multiple methods to verify cytosine methylation in the chloroplast DNA of the multicellular brown alga Saccharina japonica. Cytosine DNA methylation is a heritable process which plays important roles in regulating development throughout the life cycle of an organism. Although methylation of nuclear DNA has been studied extensively, little is known about the state and role of DNA methylation in chloroplast genomes, especially in marine algae. Here, we have applied an integrated approach encompassing whole-genome bisulfite sequencing, Methylated DNA Immunoprecipitation, gene co-expression networks and photophysiological analyses to provide evidence for the role of chloroplast DNA methylation in a marine alga, the multicellular brown alga Saccharina japonica. Although the overall methylation level was relatively low in the chloroplast genome of S. japonica, gametophytes exhibited higher methylation levels than sporophytes. Gene-specific bisulfite-cloning sequencing provided additional evidence for the methylation of key photosynthetic genes. Many of them were highly expressed in sporophytes whereas genes involved in transcription, translation and biosynthesis were strongly expressed in gametophytes. Nucleus-encoded photosynthesis genes were co-expressed with their chloroplast-encoded counterparts potentially contributing to the higher photosynthetic performance in sporophytes compared to gametophytes where these co-expression networks were less pronounced. A nucleus-encoded DNA methyltransferase of the DNMT2 family is assumed to be responsible for the methylation of the chloroplast genome because it is predicted to possess a plastid transit peptide.

  • integrative analysis of chloroplast DNA methylation in a marine alga saccharina japonica
    Plant Molecular Biology, 2021
    Co-Authors: Linhong Teng, Wentao Han, Xiao Fan, Xiaowen Zhang, Yitao Wang, Sadequr Rahman, Matteo Pellegrini, Thomas Mock
    Abstract:

    We applied an integrative approach using multiple methods to verify cytosine methylation in the chloroplast DNA of the multicellular brown alga Saccharina japonica. Cytosine DNA methylation is a heritable process which plays important roles in regulating development throughout the life cycle of an organism. Although methylation of nuclear DNA has been studied extensively, little is known about the state and role of DNA methylation in chloroplast genomes, especially in marine algae. Here, we have applied an integrated approach encompassing whole-genome bisulfite sequencing, Methylated DNA Immunoprecipitation, gene co-expression networks and photophysiological analyses to provide evidence for the role of chloroplast DNA methylation in a marine alga, the multicellular brown alga Saccharina japonica. Although the overall methylation level was relatively low in the chloroplast genome of S. japonica, gametophytes exhibited higher methylation levels than sporophytes. Gene-specific bisulfite-cloning sequencing provided additional evidence for the methylation of key photosynthetic genes. Many of them were highly expressed in sporophytes whereas genes involved in transcription, translation and biosynthesis were strongly expressed in gametophytes. Nucleus-encoded photosynthesis genes were co-expressed with their chloroplast-encoded counterparts potentially contributing to the higher photosynthetic performance in sporophytes compared to gametophytes where these co-expression networks were less pronounced. A nucleus-encoded DNA methyltransferase of the DNMT2 family is assumed to be responsible for the methylation of the chloroplast genome because it is predicted to possess a plastid transit peptide.

Philippos C Patsalis - One of the best experts on this subject based on the ideXlab platform.

  • variability of ffDNA in maternal plasma does not prevent correct classification of trisomy 21 using medip qpcr methodology
    Prenatal Diagnosis, 2013
    Co-Authors: Skevi Kyriakou, Elena Kypri, Christiana Spyrou, Evdokia Tsaliki, Voula Velissariou, Elisavet A Papageorgiou, Philippos C Patsalis
    Abstract:

    Objective The goal of this study is to evaluate the amount of free fetal DNA (ffDNA), total DNA, and ‘fetal fraction’ found in maternal plasma and whether these influence the enrichment ratios of differentially Methylated regions (DMRs) and the correct classification of trisomy 21 using the Methylated DNA Immunoprecipitation-quantitative polymerase chain reaction (MeDIP-qPCR)-based noninvasive prenatal diagnostic methodology applied in peripheral blood. Methods Absolute quantification of ffDNA using DYS14 and total DNA using β-globin was applied in 83 maternal plasma samples. The quantification values for all 83 samples were correlated with the enrichment ratios of all seven DMRs and D-values that were obtained from the diagnostic formula of MeDIP-qPCR method. Results Our analysis concluded that trisomy 21 samples had significantly higher ffDNA and total DNA levels compared with those of normal samples. Enrichment ratios of the majority of DMRs studied exhibited no association with ffDNA, total DNA, and ‘fetal fraction’, and only a small portion of DMRs exhibited moderate association. Correlation studies of ffDNA, total DNA, and fetal fraction with the diagnostic D-value showed weak to no association but without affecting the classification of trisomy 21. Conclusion Overall, the variability of ffDNA and total DNA among maternal samples does not affect the correct trisomy 21 classification using MeDIP-qPCR methodology applied in peripheral blood. © 2013 John Wiley & Sons, Ltd.

  • fetal specific DNA methylation ratio permits noninvasive prenatal diagnosis of trisomy 21
    Nature Medicine, 2011
    Co-Authors: Elisavet A Papageorgiou, Evdokia Tsaliki, Voula Velissariou, Alex Karagrigoriou, Nigel P Carter, Philippos C Patsalis
    Abstract:

    Noninvasive testing for Down's syndrome (trisomy 21) would greatly reduce the risks associated with the more invasive techniques used currently. Earlier identification of differentially Methylated regions between fetal DNA and maternal peripheral blood has now enabled Elisavet Papageorgiou and her colleagues to develop a strategy involving Methylated DNA Immunoprecipitation in combination with real-time quantitative PCR that discriminates normal from trisomy 21 cases in maternal peripheral blood with high sensitivity.

  • fetal specific DNA methylation ratio permits noninvasive prenatal diagnosis of trisomy 21
    Nature Medicine, 2011
    Co-Authors: Elisavet A Papageorgiou, Evdokia Tsaliki, Voula Velissariou, Alex Karagrigoriou, Nigel P Carter, Philippos C Patsalis
    Abstract:

    The trials performed worldwide toward noninvasive prenatal diagnosis (NIPD) of Down's syndrome (or trisomy 21) have shown the commercial and medical potential of NIPD compared to the currently used invasive prenatal diagnostic procedures. Extensive investigation of methylation differences between the mother and the fetus has led to the identification of differentially Methylated regions (DMRs). In this study, we present a strategy using the Methylated DNA Immunoprecipitation (MeDiP) methodology in combination with real-time quantitative PCR (qPCR) to achieve fetal chromosome dosage assessment, which can be performed noninvasively through the analysis of fetal-specific DMRs. We achieved noninvasive prenatal detection of trisomy 21 by determining the methylation ratio of normal and trisomy 21 cases for each tested fetal-specific DMR present in maternal peripheral blood, followed by further statistical analysis. The application of this fetal-specific methylation ratio approach provided correct diagnosis of 14 trisomy 21 and 26 normal cases.

Elisavet A Papageorgiou - One of the best experts on this subject based on the ideXlab platform.

  • variability of ffDNA in maternal plasma does not prevent correct classification of trisomy 21 using medip qpcr methodology
    Prenatal Diagnosis, 2013
    Co-Authors: Skevi Kyriakou, Elena Kypri, Christiana Spyrou, Evdokia Tsaliki, Voula Velissariou, Elisavet A Papageorgiou, Philippos C Patsalis
    Abstract:

    Objective The goal of this study is to evaluate the amount of free fetal DNA (ffDNA), total DNA, and ‘fetal fraction’ found in maternal plasma and whether these influence the enrichment ratios of differentially Methylated regions (DMRs) and the correct classification of trisomy 21 using the Methylated DNA Immunoprecipitation-quantitative polymerase chain reaction (MeDIP-qPCR)-based noninvasive prenatal diagnostic methodology applied in peripheral blood. Methods Absolute quantification of ffDNA using DYS14 and total DNA using β-globin was applied in 83 maternal plasma samples. The quantification values for all 83 samples were correlated with the enrichment ratios of all seven DMRs and D-values that were obtained from the diagnostic formula of MeDIP-qPCR method. Results Our analysis concluded that trisomy 21 samples had significantly higher ffDNA and total DNA levels compared with those of normal samples. Enrichment ratios of the majority of DMRs studied exhibited no association with ffDNA, total DNA, and ‘fetal fraction’, and only a small portion of DMRs exhibited moderate association. Correlation studies of ffDNA, total DNA, and fetal fraction with the diagnostic D-value showed weak to no association but without affecting the classification of trisomy 21. Conclusion Overall, the variability of ffDNA and total DNA among maternal samples does not affect the correct trisomy 21 classification using MeDIP-qPCR methodology applied in peripheral blood. © 2013 John Wiley & Sons, Ltd.

  • medip real time qpcr of maternal peripheral blood reliably identifies trisomy 21
    Prenatal Diagnosis, 2012
    Co-Authors: Skevi Kyriakou, Elena Kypri, Christiana Spyrou, Evdokia Tsaliki, Elisavet A Papageorgiou, George Koumbaris, Chrysovalanto Sotiriou, Evi Touvana, Anna Keravnou
    Abstract:

    Objective To reevaluate the efficiency of the 12 differentially Methylated regions (DMRs) used in the Methylated DNA Immunoprecipitation (MeDIP) real-time quantitative polymerase chain reaction (real-time qPCR) based approach, develop an improved version of the diagnostic formula and perform a larger validation study. Methods Twelve selected DMRs were checked for copy number variants in the Database of Genomic Variants. The DMRs located within copy number variants were excluded from the analysis. One hundred and seventy-five maternal peripheral blood samples were used to reconstruct and evaluate the new diagnostic formula and for a larger-scale blinded validation study using MeDIP real-time qPCR. Results Seven DMRs entered the final model of the prediction equation and a larger blinded validation study demonstrated 100% sensitivity and 99.2% specificity. No significant evidence for association was observed between cell free fetal DNA concentration and D value. Conclusion The MeDIP real-time qPCR method for noninvasive prenatal diagnosis of trisomy 21 was confirmed and revalidated in 175 samples with satisfactory results demonstrating that it is accurate and reproducible. We are currently working towards simplification of the method to make it more robust and therefore easily, accurately, and rapidly reproduced and adopted by other laboratories. Nevertheless, larger scale validation studies are necessary before the MeDIP real-time qPCR-based method could be applied in clinical practice. © 2012 John Wiley & Sons, Ltd.

  • fetal specific DNA methylation ratio permits noninvasive prenatal diagnosis of trisomy 21
    Nature Medicine, 2011
    Co-Authors: Elisavet A Papageorgiou, Evdokia Tsaliki, Voula Velissariou, Alex Karagrigoriou, Nigel P Carter, Philippos C Patsalis
    Abstract:

    Noninvasive testing for Down's syndrome (trisomy 21) would greatly reduce the risks associated with the more invasive techniques used currently. Earlier identification of differentially Methylated regions between fetal DNA and maternal peripheral blood has now enabled Elisavet Papageorgiou and her colleagues to develop a strategy involving Methylated DNA Immunoprecipitation in combination with real-time quantitative PCR that discriminates normal from trisomy 21 cases in maternal peripheral blood with high sensitivity.

  • fetal specific DNA methylation ratio permits noninvasive prenatal diagnosis of trisomy 21
    Nature Medicine, 2011
    Co-Authors: Elisavet A Papageorgiou, Evdokia Tsaliki, Voula Velissariou, Alex Karagrigoriou, Nigel P Carter, Philippos C Patsalis
    Abstract:

    The trials performed worldwide toward noninvasive prenatal diagnosis (NIPD) of Down's syndrome (or trisomy 21) have shown the commercial and medical potential of NIPD compared to the currently used invasive prenatal diagnostic procedures. Extensive investigation of methylation differences between the mother and the fetus has led to the identification of differentially Methylated regions (DMRs). In this study, we present a strategy using the Methylated DNA Immunoprecipitation (MeDiP) methodology in combination with real-time quantitative PCR (qPCR) to achieve fetal chromosome dosage assessment, which can be performed noninvasively through the analysis of fetal-specific DMRs. We achieved noninvasive prenatal detection of trisomy 21 by determining the methylation ratio of normal and trisomy 21 cases for each tested fetal-specific DMR present in maternal peripheral blood, followed by further statistical analysis. The application of this fetal-specific methylation ratio approach provided correct diagnosis of 14 trisomy 21 and 26 normal cases.