Quantitative Methods to Monitor RNA Biomarkers in Myotonic Dystrophy

Abstract Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are human neuromuscular disorders associated with mutations of simple repetitive sequences in affected genes. The abnormal expansion of CTG repeats in the 3′-UTR of the DMPK gene elicits DM1, whereas elongated CCTG repeats in intron 1 of ZNF9...

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Autores principales:	Marzena Wojciechowska, Krzysztof Sobczak, Piotr Kozlowski, Saam Sedehizadeh, Agnieszka Wojtkowiak-Szlachcic, Karol Czubak, Robert Markus, Anna Lusakowska, Anna Kaminska, J. David Brook
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2018
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/5f98b45be94545c6a92269f3ba50f1e7
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spelling	oai:doaj.org-article:5f98b45be94545c6a92269f3ba50f1e72021-12-02T15:08:40ZQuantitative Methods to Monitor RNA Biomarkers in Myotonic Dystrophy10.1038/s41598-018-24156-x2045-2322https://doaj.org/article/5f98b45be94545c6a92269f3ba50f1e72018-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-24156-xhttps://doaj.org/toc/2045-2322Abstract Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are human neuromuscular disorders associated with mutations of simple repetitive sequences in affected genes. The abnormal expansion of CTG repeats in the 3′-UTR of the DMPK gene elicits DM1, whereas elongated CCTG repeats in intron 1 of ZNF9/CNBP triggers DM2. Pathogenesis of both disorders is manifested by nuclear retention of expanded repeat-containing RNAs and aberrant alternative splicing. The precise determination of absolute numbers of mutant RNA molecules is important for a better understanding of disease complexity and for accurate evaluation of the efficacy of therapeutic drugs. We present two quantitative methods, Multiplex Ligation-Dependent Probe Amplification and droplet digital PCR, for studying the mutant DMPK transcript (DMPK expRNA) and the aberrant alternative splicing in DM1 and DM2 human tissues and cells. We demonstrate that in DM1, the DMPK expRNA is detected in higher copy number than its normal counterpart. Moreover, the absolute number of the mutant transcript indicates its low abundance with only a few copies per cell in DM1 fibroblasts. Most importantly, in conjunction with fluorescence in-situ hybridization experiments, our results suggest that in DM1 fibroblasts, the vast majority of nuclear RNA foci consist of a few molecules of DMPK expRNA.Marzena WojciechowskaKrzysztof SobczakPiotr KozlowskiSaam SedehizadehAgnieszka Wojtkowiak-SzlachcicKarol CzubakRobert MarkusAnna LusakowskaAnna KaminskaJ. David BrookNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-13 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Marzena Wojciechowska Krzysztof Sobczak Piotr Kozlowski Saam Sedehizadeh Agnieszka Wojtkowiak-Szlachcic Karol Czubak Robert Markus Anna Lusakowska Anna Kaminska J. David Brook Quantitative Methods to Monitor RNA Biomarkers in Myotonic Dystrophy
description	Abstract Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are human neuromuscular disorders associated with mutations of simple repetitive sequences in affected genes. The abnormal expansion of CTG repeats in the 3′-UTR of the DMPK gene elicits DM1, whereas elongated CCTG repeats in intron 1 of ZNF9/CNBP triggers DM2. Pathogenesis of both disorders is manifested by nuclear retention of expanded repeat-containing RNAs and aberrant alternative splicing. The precise determination of absolute numbers of mutant RNA molecules is important for a better understanding of disease complexity and for accurate evaluation of the efficacy of therapeutic drugs. We present two quantitative methods, Multiplex Ligation-Dependent Probe Amplification and droplet digital PCR, for studying the mutant DMPK transcript (DMPK expRNA) and the aberrant alternative splicing in DM1 and DM2 human tissues and cells. We demonstrate that in DM1, the DMPK expRNA is detected in higher copy number than its normal counterpart. Moreover, the absolute number of the mutant transcript indicates its low abundance with only a few copies per cell in DM1 fibroblasts. Most importantly, in conjunction with fluorescence in-situ hybridization experiments, our results suggest that in DM1 fibroblasts, the vast majority of nuclear RNA foci consist of a few molecules of DMPK expRNA.
format	article
author	Marzena Wojciechowska Krzysztof Sobczak Piotr Kozlowski Saam Sedehizadeh Agnieszka Wojtkowiak-Szlachcic Karol Czubak Robert Markus Anna Lusakowska Anna Kaminska J. David Brook
author_facet	Marzena Wojciechowska Krzysztof Sobczak Piotr Kozlowski Saam Sedehizadeh Agnieszka Wojtkowiak-Szlachcic Karol Czubak Robert Markus Anna Lusakowska Anna Kaminska J. David Brook
author_sort	Marzena Wojciechowska
title	Quantitative Methods to Monitor RNA Biomarkers in Myotonic Dystrophy
title_short	Quantitative Methods to Monitor RNA Biomarkers in Myotonic Dystrophy
title_full	Quantitative Methods to Monitor RNA Biomarkers in Myotonic Dystrophy
title_fullStr	Quantitative Methods to Monitor RNA Biomarkers in Myotonic Dystrophy
title_full_unstemmed	Quantitative Methods to Monitor RNA Biomarkers in Myotonic Dystrophy
title_sort	quantitative methods to monitor rna biomarkers in myotonic dystrophy
publisher	Nature Portfolio
publishDate	2018
url	https://doaj.org/article/5f98b45be94545c6a92269f3ba50f1e7
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_version_	1718388061535993856

Quantitative Methods to Monitor RNA Biomarkers in Myotonic Dystrophy

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