{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,18]],"date-time":"2026-04-18T20:07:45Z","timestamp":1776542865701,"version":"3.51.2"},"reference-count":32,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2021,1,21]],"date-time":"2021-01-21T00:00:00Z","timestamp":1611187200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100010661","name":"Horizon 2020 Framework Programme","doi-asserted-by":"publisher","award":["779257"],"award-info":[{"award-number":["779257"]}],"id":[{"id":"10.13039\/100010661","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002426","name":"Fondazione Telethon","doi-asserted-by":"publisher","award":["GSP15001"],"award-info":[{"award-number":["GSP15001"]}],"id":[{"id":"10.13039\/501100002426","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100013223","name":"PTC Therapeutics","doi-asserted-by":"publisher","award":["n.a."],"award-info":[{"award-number":["n.a."]}],"id":[{"id":"10.13039\/100013223","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Genes"],"abstract":"<jats:p>Next generation sequencing (NGS) has changed our approach to diagnosis of genetic disorders. Nowadays, the most comprehensive application of NGS is whole genome sequencing (WGS) that is able to detect virtually all DNA variations. However, even after accurate WGS, many genetic conditions remain unsolved. This may be due to the current NGS protocols, based on DNA fragmentation and short reads. To overcome these limitations, we applied a linked-read sequencing technology that combines single-molecule barcoding with short-read WGS. We were able to assemble haplotypes and distinguish between alleles along the genome. As an exemplary case, we studied the case of a female carrier of X-linked muscular dystrophy with an unsolved genetic status. A deletion of exons 16\u201329 in DMD gene was responsible for the disease in her family, but she showed a normal dosage of these exons by Multiplex Ligation-dependent Probe Amplification (MLPA) and array CGH. This situation is usually considered compatible with a \u201cnon-carrier\u201d status. Unexpectedly, the girl also showed an increased dosage of flanking exons 1\u201315 and 30\u201334. Using linked-read WGS, we were able to distinguish between the two X chromosomes. In the first allele, we found the 16\u201329 deletion, while the second allele showed a 1\u201334 duplication: in both cases, linked-read WGS correctly mapped the borders at single-nucleotide resolution. This duplication in trans apparently restored the normal dosage of exons 16\u201329 seen by quantitative assays. This had a dramatic impact in genetic counselling, by converting a non-carrier into a double carrier status prediction. We conclude that linked-read WGS should be considered as a valuable option to improve our understanding of unsolved genetic conditions.<\/jats:p>","DOI":"10.3390\/genes12020133","type":"journal-article","created":{"date-parts":[[2021,1,21]],"date-time":"2021-01-21T09:49:21Z","timestamp":1611222561000},"page":"133","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["Linked-Read Whole Genome Sequencing Solves a Double DMD Gene Rearrangement"],"prefix":"10.3390","volume":"12","author":[{"given":"Maria Elena","family":"Onore","sequence":"first","affiliation":[{"name":"UOSID Genetica Medica e Cardiomiologia, Dipartimento di Medicina di Precisione, Universit\u00e0 degli Studi della Campania \u201cLuigi Vanvitelli\u201d, 80138 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Annalaura","family":"Torella","sequence":"additional","affiliation":[{"name":"UOSID Genetica Medica e Cardiomiologia, Dipartimento di Medicina di Precisione, Universit\u00e0 degli Studi della Campania \u201cLuigi Vanvitelli\u201d, 80138 Napoli, Italy"},{"name":"Telethon Institute of Genetics and Medicine, 80078 Pozzuoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9440-1080","authenticated-orcid":false,"given":"Francesco","family":"Musacchia","sequence":"additional","affiliation":[{"name":"Telethon Institute of Genetics and Medicine, 80078 Pozzuoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Paola","family":"D\u2019Ambrosio","sequence":"additional","affiliation":[{"name":"UOSID Genetica Medica e Cardiomiologia, Dipartimento di Medicina di Precisione, Universit\u00e0 degli Studi della Campania \u201cLuigi Vanvitelli\u201d, 80138 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3750-1592","authenticated-orcid":false,"given":"Mariateresa","family":"Zanobio","sequence":"additional","affiliation":[{"name":"UOSID Genetica Medica e Cardiomiologia, Dipartimento di Medicina di Precisione, Universit\u00e0 degli Studi della Campania \u201cLuigi Vanvitelli\u201d, 80138 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9454-2964","authenticated-orcid":false,"given":"Francesca","family":"Del Vecchio Blanco","sequence":"additional","affiliation":[{"name":"UOSID Genetica Medica e Cardiomiologia, Dipartimento di Medicina di Precisione, Universit\u00e0 degli Studi della Campania \u201cLuigi Vanvitelli\u201d, 80138 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9418-125X","authenticated-orcid":false,"given":"Giulio","family":"Piluso","sequence":"additional","affiliation":[{"name":"UOSID Genetica Medica e Cardiomiologia, Dipartimento di Medicina di Precisione, Universit\u00e0 degli Studi della Campania \u201cLuigi Vanvitelli\u201d, 80138 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vincenzo","family":"Nigro","sequence":"additional","affiliation":[{"name":"UOSID Genetica Medica e Cardiomiologia, Dipartimento di Medicina di Precisione, Universit\u00e0 degli Studi della Campania \u201cLuigi Vanvitelli\u201d, 80138 Napoli, Italy"},{"name":"Telethon Institute of Genetics and Medicine, 80078 Pozzuoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,1,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"731","DOI":"10.1016\/S1474-4422(03)00585-4","article-title":"Dystrophin and mutations: One gene, several proteins, multiple phenotypes","volume":"2","author":"Muntoni","year":"2003","journal-title":"Lancet Neurol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"65","DOI":"10.2353\/jmoldx.2010.090074","article-title":"One Hundred Twenty-One Dystrophin Point Mutations Detected from Stored DNA Samples by Combinatorial Denaturing High-Performance Liquid Chromatography","volume":"12","author":"Torella","year":"2010","journal-title":"J. Mol. Diagn."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Torella, A., Zanobio, M., Zeuli, R., Del Vecchio Blanco, F., Savarese, M., Giugliano, T., Garofalo, A., Piluso, G., Politano, L., and Nigro, V. (2020). The position of nonsense mutations can predict the phenotype severity: A survey on the DMD gene. PLoS ONE, 15.","DOI":"10.1371\/journal.pone.0237803"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1002\/mgg3.108","article-title":"Complex genomic rearrangements in the dystrophin gene due to replication-based mechanisms","volume":"2","author":"Baskin","year":"2014","journal-title":"Mol. Genet. Genom. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"631","DOI":"10.1016\/j.nmd.2017.04.003","article-title":"Deep intronic variants introduce DMD pseudoexon in patient with muscular dystrophy","volume":"27","author":"Zaum","year":"2017","journal-title":"Neuromuscul. Disord."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"973","DOI":"10.1373\/clinchem.2007.097881","article-title":"Log-PCR: A New Tool for Immediate and Cost-Effective Diagnosis of up to 85% of Dystrophin Gene Mutations","volume":"54","author":"Trimarco","year":"2008","journal-title":"Clin. Chem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1231","DOI":"10.1038\/sj.ejhg.5201465","article-title":"Deletion and duplication screening in the DMD gene using MLPA","volume":"13","author":"Lalic","year":"2005","journal-title":"Eur. J. Hum. Genet."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"18168","DOI":"10.3390\/molecules201018168","article-title":"Duchenne Muscular Dystrophy: From Diagnosis to Therapy","volume":"20","author":"Falzarano","year":"2015","journal-title":"Molecules"},{"key":"ref_9","first-page":"196","article-title":"Next generation sequencing (NGS) strategies for the genetic testing of myopathies","volume":"31","author":"Nigro","year":"2012","journal-title":"Acta Myol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1212\/WNL.0000000000002800","article-title":"The genetic basis of undiagnosed muscular dystrophies and myopathies: Results from 504 patients","volume":"87","author":"Savarese","year":"2016","journal-title":"Neurology"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1016\/S1474-4422(18)30024-3","article-title":"Diagnosis and management of Duchenne muscular dystrophy, part 1: Diagnosis, and neuromuscular, rehabilitation, endocrine, and gastrointestinal and nutritional management","volume":"17","author":"Birnkrant","year":"2018","journal-title":"Lancet Neurol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"435","DOI":"10.1038\/gim.2017.119","article-title":"Improved diagnostic yield compared with targeted gene sequencing panels suggests a role for whole-genome sequencing as a first-tier genetic test","volume":"20","author":"Lionel","year":"2018","journal-title":"Genet. Med."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"635","DOI":"10.1101\/gr.234443.118","article-title":"Resolving the full spectrum of human genome variation using Linked-Reads","volume":"29","author":"Marks","year":"2019","journal-title":"Genome Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"e1114","DOI":"10.1002\/mgg3.1114","article-title":"Genome sequencing in cytogenetics: Comparison of short-read and linked-read approaches for germline structural variant detection and characterization","volume":"8","author":"Uguen","year":"2020","journal-title":"Mol. Genet. Genom. Med."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2041","DOI":"10.1002\/acn3.51201","article-title":"Long-read whole-genome sequencing for the genetic diagnosis of dystrophinopathies","volume":"7","author":"Xie","year":"2020","journal-title":"Ann. Clin. Transl. Neurol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"477","DOI":"10.3233\/JND-200523","article-title":"Identification and Characterization of Splicing Defects by Single-Molecule Real-Time Sequencing Technology (PacBio)","volume":"7","author":"Savarese","year":"2020","journal-title":"J. Neuromuscul. Dis."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1038\/gim.2017.86","article-title":"Long-read genome sequencing identifies causal structural variation in a Mendelian disease","volume":"20","author":"Merker","year":"2018","journal-title":"Genet. Med."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Ott, A., Schnable, J.C., Yeh, C.T., Wu, L., Liu, C., Hu, H.C., Dalgard, C.L., Sarkar, S., and Schnable, P.S. (2018). Linked read technology for assembling large complex and polyploid genomes. BMC Genom., 19.","DOI":"10.1186\/s12864-018-5040-z"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Giugliano, T., Savarese, M., Garofalo, A., Picillo, E., Fiorillo, C., D\u2019Amico, A., Maggi, L., Ruggiero, L., Vercelli, L., and Magri, F. (2018). Copy Number Variants Account for a Tiny Fraction of Undiagnosed Myopathic Patients. Genes, 9.","DOI":"10.3390\/genes9110524"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Savarese, M., Piluso, G., Orteschi, D., Di Fruscio, G., Dionisi, M., Blanco, F.D.V., Torella, A., Giugliano, T., Iacomino, M., and Zollino, M. (2012). Enhancer Chip: Detecting Human Copy Number Variations in Regulatory Elements. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0052264"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"353","DOI":"10.1093\/bioinformatics\/btx712","article-title":"Identifying structural variants using linked-read sequencing data","volume":"34","author":"Elyanow","year":"2018","journal-title":"Bioinformatics"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1336","DOI":"10.1101\/gr.077065.108","article-title":"An MCMC algorithm for haplotype assembly from whole-genome sequence data","volume":"18","author":"Bansal","year":"2008","journal-title":"Genome Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1584","DOI":"10.1373\/clinchem.2011.168898","article-title":"Motor Chip: A Comparative Genomic Hybridization Microarray for Copy-Number Mutations in 245 Neuromuscular Disorders","volume":"57","author":"Piluso","year":"2011","journal-title":"Clin. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1534\/genetics.115.183483","article-title":"Genome-Wide Structural Variation Detection by Genome Mapping on Nanochannel Arrays","volume":"202","author":"Mak","year":"2016","journal-title":"Genetics"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"224","DOI":"10.1038\/nrg.2015.25","article-title":"Mechanisms underlying structural variant formation in genomic disorders","volume":"17","author":"Carvalho","year":"2016","journal-title":"Nat. Rev. Genet."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1055\/s-0039-1688446","article-title":"Next-Generation Sequencing and Emerging Technologies","volume":"45","author":"Kumar","year":"2019","journal-title":"Semin. Thromb. Hemost."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"278","DOI":"10.1016\/j.gpb.2015.08.002","article-title":"PacBio Sequencing and Its Applications","volume":"13","author":"Rhoads","year":"2015","journal-title":"Genom. Proteom. Bioinform."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1016\/j.gpb.2016.05.004","article-title":"Oxford Nanopore MinION Sequencing and Genome Assembly","volume":"14","author":"Lu","year":"2016","journal-title":"Genom. Proteom. Bioinform."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1038\/s10038-019-0671-8","article-title":"Long-read sequencing for rare human genetic diseases","volume":"65","author":"Mitsuhashi","year":"2020","journal-title":"J. Hum. Genet."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"5585","DOI":"10.1038\/s41467-019-13397-7","article-title":"LinkedSV for detection of mosaic structural variants from linked-read exome and genome sequencing data","volume":"10","author":"Fang","year":"2019","journal-title":"Nat. Commun."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"4397","DOI":"10.1093\/bioinformatics\/btz239","article-title":"Structural variant analysis for linked-read sequencing data with gemtools","volume":"35","author":"Greer","year":"2019","journal-title":"Bioinformatics"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"921","DOI":"10.1056\/NEJM199309233291304","article-title":"Brief report: Deletion of the Dystrophin Muscle-Promoter Region Associated with X-Linked Dilated Cardiomyopathy","volume":"329","author":"Muntoni","year":"1993","journal-title":"N. Engl. J. Med."}],"container-title":["Genes"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4425\/12\/2\/133\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T05:13:22Z","timestamp":1760159602000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4425\/12\/2\/133"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,1,21]]},"references-count":32,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2021,2]]}},"alternative-id":["genes12020133"],"URL":"https:\/\/doi.org\/10.3390\/genes12020133","relation":{},"ISSN":["2073-4425"],"issn-type":[{"value":"2073-4425","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,1,21]]}}}