Pathogenic large inversions are rarely reported on DMD gene due to the lack of effective detection methods. Here we report two DMD pedigrees and proposed a reliable pipeline to define large inversions in DMD patients. In the first pedigree, conventional approaches including multiplex ligation-dependent probe amplification, and whole-exome sequencing by next generation sequencing were failed to detect any pathologic variant. Then an advanced analysis pipeline which consists of RNA-seq, cDNA array capture sequencing, optical mapping, long-read sequencing was built. RNA-seq and cDNA capture sequencing showed a complete absence of transcripts of exons 3–55. Optical mapping identified a 55 Mb pericentric inversion between Xp21 and Xq21. Subsequently, long-read sequencing and Sanger sequencing determined the inversion breakpoints at 32,915,769 and 87,989,324 of the X chromosomes. In the second pedigree, long-read sequencing was directly conducted and Sanger sequencing was performed to verify the mutation. Long-read sequencing and Sanger sequencing found breakpoints at 32,581,576 and 127,797,236 on DMD gene directly. In conclusion, large inversion might be a rare but important mutation type in DMD gene. An effective pipeline was built in detecting large inversion mutations based on long-read sequencing platforms.
This is a preview of subscription content, access via your institution
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
The data are available from the corresponding author on reasonable request.
Burghes AH, Logan C, Hu X, Belfall B, Worton RG, Ray PN. A cDNA clone from the Duchenne/Becker muscular dystrophy gene. Nature. 1987;328:434–7.
Kunkel LM, Monaco AP, Middlesworth W, Ochs HD, Latt SA. Specific cloning of DNA fragments absent from the DNA of a male patient with an X chromosome deletion. Proc Natl Acad Sci USA. 1985;82:4778–82.
Emery AE. The muscular dystrophies. Lancet 2002;359:687–95.
Aartsma‐Rus A, Van Deutekom JC, Fokkema IF, Van Ommen GJB, Den Dunnen JT. Entries in the Leiden Duchenne muscular dystrophy mutation database: An overview of mutation types and paradoxical cases that confirm the reading‐frame rule. Muscle Nerve: Off J Am Assoc Electrodiagn Med. 2006;34:135–44.
Rybakova IN, Patel JR, Ervasti JM. The dystrophin complex forms a mechanically strong link between the sarcolemma and costameric actin. J Cell Biol. 2000;150:1209–14.
Koenig M, Beggs A, Moyer M, Scherpf S, Heindrich K, Bettecken T, et al. The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion. Am J Hum Genet. 1989;45:498.
Tuffery‐Giraud S, Béroud C, Leturcq F, Yaou RB, Hamroun D, Michel‐Calemard L, et al. Genotype–phenotype analysis in 2,405 patients with a dystrophinopathy using the UMD–DMD database: a model of nationwide knowledgebase. Hum Mutat. 2009;30:934–45.
Bladen CL, Salgado D, Monges S, Foncuberta ME, Kekou K, Kosma K, et al. The TREAT‐NMD DMD Global Database: analysis of more than 7,000 Duchenne muscular dystrophy mutations. Hum Mutat. 2015;36:395–402.
Tong YR, Geng C, Guan YZ, Zhao YH, Ren HT, Yao FX, et al. A Comprehensive Analysis of 2013 Dystrophinopathies in China: A Report From National Rare Disease Center. Front Neurol. 2020;11:572006.
Trippe H, Wieczorek S, Kötting J, Kress W, Schara U. Xp21/A translocation: a rarely considered genetic cause for manifesting carriers of duchenne muscular dystrophy. Neuropediatrics 2014;45:333–5.
Ligon AH, Kashork CD, Richards CS, Shaffer LG. Identification of female carriers for Duchenne and Becker muscular dystrophies using a FISH-based approach. Eur J Hum Genet. 2000;8:293–8.
Ishmukhametova A, Van Kien PK, Méchin D, Thorel D, Vincent M-C, Rivier F, et al. Comprehensive oligonucleotide array-comparative genomic hybridization analysis: new insights into the molecular pathology of the DMD gene. Eur J Hum Genet. 2012;20:1096–100.
van Dijk EL, Jaszczyszyn Y, Naquin D, Thermes C. The Third Revolution in Sequencing Technology. Trends Genet. 2018;34:666–81.
Zhang Q, Xu X, Ding L, Li H, Xu C, Gong Y, et al. Clinical application of single‐molecule optical mapping to a multigeneration FSHD1 pedigree. Mol Genet Genom Med. 2019;7:e565.
Roberts RJ, Carneiro MO, Schatz MC. The advantages of SMRT sequencing. Genome Biol. 2013;14:405.
Loman NJ, Quick J, Simpson JT. A complete bacterial genome assembled de novo using only nanopore sequencing data. Nat Methods. 2015;12:733–5.
Barseghyan H, Tang W, Wang RT, Almalvez M, Segura E, Bramble MS, et al. Next-generation mapping: a novel approach for detection of pathogenic structural variants with a potential utility in clinical diagnosis. Genome Med. 2017;9:90.
Mantere T, Kersten S, Hoischen A. Long-read sequencing emerging in medical genetics. Front Genet. 2019;10:426.
Chaisson MJ, Huddleston J, Dennis MY, Sudmant PH, Malig M, Hormozdiari F, et al. Resolving the complexity of the human genome using single-molecule sequencing. Nature 2015;517:608–11.
Sanchis-Juan A, Stephens J, French CE, Gleadall N, Mégy K, Penkett C, et al. Complex structural variants in Mendelian disorders: identification and breakpoint resolution using short-and long-read genome sequencing. Genome Med. 2018;10:95.
Collins RL, Brand H, Redin CE, Hanscom C, Antolik C, Stone MR, et al. Defining the diverse spectrum of inversions, complex structural variation, and chromothripsis in the morbid human genome. Genome Biol. 2017;18:36.
Gonorazky H, Liang M, Cummings B, Lek M, Micallef J, Hawkins C, et al. RNA seq analysis for the diagnosis of muscular dystrophy. Ann Clin Transl Neurol. 2016;3:55–60.
Dai Y, Li P, Wang Z, Liang F, Yang F, Fang L, et al. Single-molecule optical mapping enables quantitative measurement of D4Z4 repeats in facioscapulohumeral muscular dystrophy (FSHD). J Med Genet. 2020;57:109–20.
Deng J, Yu J, Li P, Luan X, Cao L, Zhao J, et al. Expansion of GGC Repeat in GIPC1 Is Associated with Oculopharyngodistal Myopathy. Am J Hum Genet. 2020;106:793–804.
Okubo M, Minami N, Goto K, Goto Y, Noguchi S, Mitsuhashi S, et al. Genetic diagnosis of Duchenne/Becker muscular dystrophy using next-generation sequencing: validation analysis of DMD mutations. J Hum Genet. 2016;61:483–9.
Lim BC, Lee S, Shin JY, Kim JI, Hwang H, Kim KJ, et al. Genetic diagnosis of Duchenne and Becker muscular dystrophy using next-generation sequencing technology: comprehensive mutational search in a single platform. J Med Genet. 2011;48:731–6.
Falzarano MS, Grilli A, Zia S, Fang M, Rossi R, Gualandi F, et al. RNA-seq in DMD urinary stem cells recognized muscle-related transcription signatures and addressed the identification of atypical mutations by whole-genome sequencing. HGG Adv. 2022;3:100054.
Weckselblatt B, Rudd MK. Human structural variation: mechanisms of chromosome rearrangements. Trends Genet. 2015;31:587–99.
Carvalho CM, Ramocki MB, Pehlivan D, Franco LM, Gonzaga-Jauregui C, Fang P, et al. Inverted genomic segments and complex triplication rearrangements are mediated by inverted repeats in the human genome. Nat Genet. 2011;43:1074.
Fratter C, Dalgleish R, Allen SK, Santos R, Abbs S, Tuffery-Giraud S, et al. EMQN best practice guidelines for genetic testing in dystrophinopathies. Eur J Hum Genet. 2020;28:1141–59.
The authors express sincere gratitude to the patient and the family members who participated in the study. Special acknowledgements go to Dr Xiaoming (BGI Clinical Laboratory, Wuhan, China) for assistance for muscle tissue mRNA sequencing and to Ms Minming Zheng (Grandomics Biosciences, Beijing, China) for preparing the figures.
This study was funded by CAMS Innovation Fund for Medical Sciences (CIFMS) (No.2016-I2M-1-002) and Chinese National Basic Research Program (973) (No.2017YFC1001902).
The authors declare no competing interests.
This study was approved by the ethics committee of the Peking Union Medical College Hospital (IRB #JS-1233). All participants and the parents of those <18years of age at the time of genetic testing gave written informed consent.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Geng, C., Zhang, C., Li, P. et al. Identification and characterization of two DMD pedigrees with large inversion mutations based on a long-read sequencing pipeline. Eur J Hum Genet (2022). https://doi.org/10.1038/s41431-022-01190-y