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WGS and RNA Studies Diagnose Noncoding DMD Variants in Males With High Creatine Kinase

OBJECTIVE: To describe the diagnostic utility of whole-genome sequencing and RNA studies in boys with suspected dystrophinopathy, for whom multiplex ligation-dependent probe amplification and exomic parallel sequencing failed to yield a genetic diagnosis, and to use remnant normal DMD splicing in 3...

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Autores principales: Waddell, Leigh B., Bryen, Samantha J., Cummings, Beryl B., Bournazos, Adam, Evesson, Frances J., Joshi, Himanshu, Marshall, Jamie L., Tukiainen, Taru, Valkanas, Elise, Weisburd, Ben, Sadedin, Simon, Davis, Mark R., Faiz, Fathimath, Gooding, Rebecca, Sandaradura, Sarah A., O'Grady, Gina L., Tchan, Michel C., Mowat, David R., Oates, Emily C., Farrar, Michelle A., Sampaio, Hugo, Ma, Alan, Neas, Katherine, Wang, Min-Xia, Charlton, Amanda, Chan, Charles, Kenwright, Diane N., Graf, Nicole, Arbuckle, Susan, Clarke, Nigel F., MacArthur, Daniel G., Jones, Kristi J., Lek, Monkol, Cooper, Sandra T.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Wolters Kluwer 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8105888/
https://www.ncbi.nlm.nih.gov/pubmed/33977140
http://dx.doi.org/10.1212/NXG.0000000000000554
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author Waddell, Leigh B.
Bryen, Samantha J.
Cummings, Beryl B.
Bournazos, Adam
Evesson, Frances J.
Joshi, Himanshu
Marshall, Jamie L.
Tukiainen, Taru
Valkanas, Elise
Weisburd, Ben
Sadedin, Simon
Davis, Mark R.
Faiz, Fathimath
Gooding, Rebecca
Sandaradura, Sarah A.
O'Grady, Gina L.
Tchan, Michel C.
Mowat, David R.
Oates, Emily C.
Farrar, Michelle A.
Sampaio, Hugo
Ma, Alan
Neas, Katherine
Wang, Min-Xia
Charlton, Amanda
Chan, Charles
Kenwright, Diane N.
Graf, Nicole
Arbuckle, Susan
Clarke, Nigel F.
MacArthur, Daniel G.
Jones, Kristi J.
Lek, Monkol
Cooper, Sandra T.
author_facet Waddell, Leigh B.
Bryen, Samantha J.
Cummings, Beryl B.
Bournazos, Adam
Evesson, Frances J.
Joshi, Himanshu
Marshall, Jamie L.
Tukiainen, Taru
Valkanas, Elise
Weisburd, Ben
Sadedin, Simon
Davis, Mark R.
Faiz, Fathimath
Gooding, Rebecca
Sandaradura, Sarah A.
O'Grady, Gina L.
Tchan, Michel C.
Mowat, David R.
Oates, Emily C.
Farrar, Michelle A.
Sampaio, Hugo
Ma, Alan
Neas, Katherine
Wang, Min-Xia
Charlton, Amanda
Chan, Charles
Kenwright, Diane N.
Graf, Nicole
Arbuckle, Susan
Clarke, Nigel F.
MacArthur, Daniel G.
Jones, Kristi J.
Lek, Monkol
Cooper, Sandra T.
author_sort Waddell, Leigh B.
collection PubMed
description OBJECTIVE: To describe the diagnostic utility of whole-genome sequencing and RNA studies in boys with suspected dystrophinopathy, for whom multiplex ligation-dependent probe amplification and exomic parallel sequencing failed to yield a genetic diagnosis, and to use remnant normal DMD splicing in 3 families to define critical levels of wild-type dystrophin bridging clinical spectrums of Duchenne to myalgia. METHODS: Exome, genome, and/or muscle RNA sequencing was performed for 7 males with elevated creatine kinase. PCR of muscle-derived complementary DNA (cDNA) studied consequences for DMD premessenger RNA (pre-mRNA) splicing. Quantitative Western blot was used to determine levels of dystrophin, relative to control muscle. RESULTS: Splice-altering intronic single nucleotide variants or structural rearrangements in DMD were identified in all 7 families. Four individuals, with abnormal splicing causing a premature stop codon and nonsense-mediated decay, expressed remnant levels of normally spliced DMD mRNA. Quantitative Western blot enabled correlation of wild-type dystrophin and clinical severity, with 0%–5% dystrophin conferring a Duchenne phenotype, 10% ± 2% a Becker phenotype, and 15% ± 2% dystrophin associated with myalgia without manifesting weakness. CONCLUSIONS: Whole-genome sequencing relied heavily on RNA studies to identify DMD splice-altering variants. Short-read RNA sequencing was regularly confounded by the effectiveness of nonsense-mediated mRNA decay and low read depth of the giant DMD mRNA. PCR of muscle cDNA provided a simple, yet informative approach. Highly relevant to genetic therapies for dystrophinopathies, our data align strongly with previous studies of mutant dystrophin in Becker muscular dystrophy, with the collective conclusion that a fractional increase in levels of normal dystrophin between 5% and 20% is clinically significant.
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spelling pubmed-81058882021-05-10 WGS and RNA Studies Diagnose Noncoding DMD Variants in Males With High Creatine Kinase Waddell, Leigh B. Bryen, Samantha J. Cummings, Beryl B. Bournazos, Adam Evesson, Frances J. Joshi, Himanshu Marshall, Jamie L. Tukiainen, Taru Valkanas, Elise Weisburd, Ben Sadedin, Simon Davis, Mark R. Faiz, Fathimath Gooding, Rebecca Sandaradura, Sarah A. O'Grady, Gina L. Tchan, Michel C. Mowat, David R. Oates, Emily C. Farrar, Michelle A. Sampaio, Hugo Ma, Alan Neas, Katherine Wang, Min-Xia Charlton, Amanda Chan, Charles Kenwright, Diane N. Graf, Nicole Arbuckle, Susan Clarke, Nigel F. MacArthur, Daniel G. Jones, Kristi J. Lek, Monkol Cooper, Sandra T. Neurol Genet Article OBJECTIVE: To describe the diagnostic utility of whole-genome sequencing and RNA studies in boys with suspected dystrophinopathy, for whom multiplex ligation-dependent probe amplification and exomic parallel sequencing failed to yield a genetic diagnosis, and to use remnant normal DMD splicing in 3 families to define critical levels of wild-type dystrophin bridging clinical spectrums of Duchenne to myalgia. METHODS: Exome, genome, and/or muscle RNA sequencing was performed for 7 males with elevated creatine kinase. PCR of muscle-derived complementary DNA (cDNA) studied consequences for DMD premessenger RNA (pre-mRNA) splicing. Quantitative Western blot was used to determine levels of dystrophin, relative to control muscle. RESULTS: Splice-altering intronic single nucleotide variants or structural rearrangements in DMD were identified in all 7 families. Four individuals, with abnormal splicing causing a premature stop codon and nonsense-mediated decay, expressed remnant levels of normally spliced DMD mRNA. Quantitative Western blot enabled correlation of wild-type dystrophin and clinical severity, with 0%–5% dystrophin conferring a Duchenne phenotype, 10% ± 2% a Becker phenotype, and 15% ± 2% dystrophin associated with myalgia without manifesting weakness. CONCLUSIONS: Whole-genome sequencing relied heavily on RNA studies to identify DMD splice-altering variants. Short-read RNA sequencing was regularly confounded by the effectiveness of nonsense-mediated mRNA decay and low read depth of the giant DMD mRNA. PCR of muscle cDNA provided a simple, yet informative approach. Highly relevant to genetic therapies for dystrophinopathies, our data align strongly with previous studies of mutant dystrophin in Becker muscular dystrophy, with the collective conclusion that a fractional increase in levels of normal dystrophin between 5% and 20% is clinically significant. Wolters Kluwer 2021-01-29 /pmc/articles/PMC8105888/ /pubmed/33977140 http://dx.doi.org/10.1212/NXG.0000000000000554 Text en Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND) (https://creativecommons.org/licenses/by-nc-nd/4.0/) , which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.
spellingShingle Article
Waddell, Leigh B.
Bryen, Samantha J.
Cummings, Beryl B.
Bournazos, Adam
Evesson, Frances J.
Joshi, Himanshu
Marshall, Jamie L.
Tukiainen, Taru
Valkanas, Elise
Weisburd, Ben
Sadedin, Simon
Davis, Mark R.
Faiz, Fathimath
Gooding, Rebecca
Sandaradura, Sarah A.
O'Grady, Gina L.
Tchan, Michel C.
Mowat, David R.
Oates, Emily C.
Farrar, Michelle A.
Sampaio, Hugo
Ma, Alan
Neas, Katherine
Wang, Min-Xia
Charlton, Amanda
Chan, Charles
Kenwright, Diane N.
Graf, Nicole
Arbuckle, Susan
Clarke, Nigel F.
MacArthur, Daniel G.
Jones, Kristi J.
Lek, Monkol
Cooper, Sandra T.
WGS and RNA Studies Diagnose Noncoding DMD Variants in Males With High Creatine Kinase
title WGS and RNA Studies Diagnose Noncoding DMD Variants in Males With High Creatine Kinase
title_full WGS and RNA Studies Diagnose Noncoding DMD Variants in Males With High Creatine Kinase
title_fullStr WGS and RNA Studies Diagnose Noncoding DMD Variants in Males With High Creatine Kinase
title_full_unstemmed WGS and RNA Studies Diagnose Noncoding DMD Variants in Males With High Creatine Kinase
title_short WGS and RNA Studies Diagnose Noncoding DMD Variants in Males With High Creatine Kinase
title_sort wgs and rna studies diagnose noncoding dmd variants in males with high creatine kinase
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8105888/
https://www.ncbi.nlm.nih.gov/pubmed/33977140
http://dx.doi.org/10.1212/NXG.0000000000000554
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