Identifying inversions with Breakpoints in the Dystrophin Gene through Long-Read Sequencing: Report of Two Cases

Identifying inversions with Breakpoints in the Dystrophin Gene through Long-Read Sequencing: Report of Two Cases | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Case Report Identifying inversions with Breakpoints in the Dystrophin Gene through Long-Read Sequencing: Report of Two Cases Liqing Chen, Xiaoping Luo, Hongling Wang, Yu Tian, Yan Liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3982190/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Sep, 2024 Read the published version in BMC Medical Genomics → Version 1 posted 4 You are reading this latest preprint version Abstract Background: Duchenne Muscular Dystrophy (DMD) is an X-linked disorder caused by mutations in the DMD gene, with large deletions being the most frequent type of mutation. Large inversions involving the DMD gene are a less common cause of the disorder, primarily because they often elude detection by standard diagnostic methods such as multiplex ligation probe amplification (MLPA) and whole exome sequencing (WES) utilizing next-generation sequencing (NGS) technologies. Case presentation : Our research uncovered two intrachromosomal inversions involved the dystrophin gene in two unrelated families through Long-read sequencing (LRS). To confirm these variants, Sanger sequencing subsequently carried out. The first case involved a pericentric inversion from DMD intron 47 to the Xq27.3. The second case featured a paracentric inversion between DMD intron 42 and Xp21.1, inherited from the mother. In both cases, simple repeat sequences (SRS) were present at the breakpoints of these inversions. Conclusions : Our findings demonstrate that LRS can be effectively used to detect atypical mutation. The identification of SRS at breakpoints in DMD patients assists in acquiring a more profound understanding of the mechanisms involved in structural variations, thereby facilitating exploration into potential treatments. DMD inversion breakpoint long-read sequencing simple repeated sequences Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background The dystrophin gene ( DMD ; MIM #300377), situated within a 2.4 Mb region on the X chromosome at the Xp21.2 locus, demonstrates complexity by producing multiple isoforms across a range of tissues. Mutations in this gene can lead to the absence of functional dystrophin protein, primarily causing progressive degradation of muscle tissue, including skeletal, myocardial, and smooth muscle. This also can result in orthopedic and respiratory complications [ 1 ]. Loss-of-function mutations in the dystrophin gene are responsible for Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD) and X-linked dilated cardiomyopathy. These mutations cover a wide spectrum of alterations, including deletions, duplications, single nucleotide variants (SNVs), insertions and complex rearrangements [ 2 ]. Among them, intragenic deletions affecting one or more exons are the most common, making up roughly 60% of all dystrophin mutations, with duplications being less frequent, appearing at a rate of 6 to 8% [ 3 ]. Complex rearrangements, like balanced translocations or inversions, are exceptionally uncommon. In investigating causative mutations in DMD/BMD patients, various methods have been utilized. Multiplex ligation-dependent probe amplification (MLPA) is the conventional method used in genetic testing to detect exonic deletions and duplications. In case where this analysis does not reveal a pathogenic mutation, the next step is to perform whole exome sequencing (WES) using next-generation sequencing (NGS) to analyze the complete coding region of the DMD gene [ 4 ]. The diagnostic rate can exceed 95% [ 5 ]. The undetected mutations that remain are primarily deep intronic mutations [ 6 ]. These can be identified through RNA analysis of muscle tissue using RT-PCR or short-read sequencing. Nonetheless, a subset of patients remains undiagnosed genetically, even with the application of these techniques, owing to the existence of exceptionally rare and complex structural variants. A previous study demonstrated that the captured long-read sequencing (LRS) panel effectively detected a spectrum of DMD mutations, ranging from SNVs to structural variations [ 7 ]. LRS, exemplified by the Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PB) single-molecule real-time sequencing (SMRT) platforms, facilitated a more precise identification of breakpoint positions for structural variations in the DMD gene. The precision in identifying breakpoints enables the decoding of the entire sequence within the DMD rearrangement region. Because the errors in the sequencing data obtained through PB sequencing were more consistent or aligned with each other, making PB easier to identify the correct sequence than ONT. In this study, we identified two intrachromosomal inversions involving the dystrophin gene within two unrelated families using LRS facilitated by the PB SMRT platform. In both cases, simple repeat sequences were observed at the breakpoints of these inversions. Case presentation Patient 1 A ten-year-old boy was admitted to our hospital due to muscle weakness. He is the elder of two brothers born to healthy, non-consanguineous Chinese parents with no significant personal or family medical history. The patient achieved independent walking at the age of fourteen months. At the age of five, he began to experience a gradual decline in his walking abilities, becoming prone to falls. Currently, he experiences difficulties in jumping and getting up after squatting. Moreover, he exhibits with intellectual disability and faces challenges in his learning at school, leading to lower grades in his examinations. His physical examination revealed hypertrophy of the gastrocnemius muscle, a positive Gowers’ sign and a waddling gait. His head circumference measured 50cm and his tendon reflexes showed weakness. Based on the provided information, we initially diagnosed dystrophinopathy clinically. A serum creatine kinase test was conducted, revealing a significantly elevated level of 10961U/L (normal ≤ 190U/L). The Wechsler Child Intelligence Scale assessed his overall IQ at 65. The electrocardiography results fell within the normal range, but the color Doppler echocardiography examination indicated left ventricular enlargement. Following the clinical diagnosis of dystrophinopathy, the patient underwent standard steroid treatment (prednisone 0,75mg/kg/d) and received regular followed-up. Genetic tests were initiated subsequently. Initially, we conducted MLPA, which did not indicate any deletions or duplication. Subsequently, we performed targeted WES using NGS to identify SNVs or splicing mutations across all 79 exons of the DMD gene. Unfortunately, no causative genetic mutations were identified through these approaches. Based on previous reports, the patient then underwent LRS directly [ 2 , 7 ]. A significant pericentric inversion, spanning 110.81Mb and mapping between chrXp21.1 and chrXq27.3, was identified through LRS. The inversion breakpoints were precisely located at the genomic positions of chrX:31,927,491 and chrX:142,742,006. Notably, the breakpoint at chrX:31,927,491 is situated within intron 47 of the DMD gene. Furthermore, the patient’s DNA revealed a 9bp deletion spanning chrX:31,927,482 − 31,927,490 (CTTTGGGAA) and a 313bp duplication within chrX:142,741,694 − 142,742,006 (Fig. 1 ). The mutation identified through LRS was subsequently confirmed by PCR/Sanger sequencing. PCR/Sanger sequencing is a widely used sequencing technology employed to validate and accurately determine the breakpoint location in inversion mutations. Two primers were designed based on the LRS results, and their specific details can be found in Fig. 2 . The inversion breakpoints were accurately determined through Sanger sequencing to be at chrX:31,927,481/142,742,828 and chrX:31,927,491/142,741,748, denoting an inversion between these genomic coordinates on the X chromosome. Additionally, a 9bp deletion at chrX:31,927,482 − 31,927,490 and an 81bp duplication at chrX:142,741,748 − 142,741,828 were identified at these breakpoints, as illustrated in Fig. 3 . Detailed analysis of the breakpoints uncovered the presence of LTR sequences at chrX:31,927,411 − 31,927,629 (219bp) and (TA)n sequences at chrX:142,741,289 − 142,741,461 (173bp), chrX142,741,503 − 142,741,649 (147bp), chrX142,741,665 − 142,741,824 (160bp) and chrX:142,741,855 − 142,742,166 (312bp) respectively. The presence of these simple repetitive sequences surrounds both breakpoints, resulting in duplicate alignments during the alignment process. Consequently, this can lead to differences between the results of long-read sequencing and Sanger sequencing. Patient 2 The second case concerns a 6-year-old boy who was admitted to our hospital presenting with abnormal gait. A confirmed family history revealed that similar conditions had afflicted his maternal uncle and cousin, both of whom exhibited comparable symptoms and sadly succumbed to their illness after reaching the age of 10 (Fig. 4 ). The boy took his first steps at 18 months. At the age of 3, he was hospitalized for a fever at a different institution, where tests revealed an abnormally high serum creatine kinase level (CK = 17215U/L, normal range: 24-204U/L). Despite these findings, he did not display any substantial motor deficits at that juncture. To establish a definitive diagnosis, he was subjected to MLPA and WES to identify common deletions and duplications within the DMD gene. These tests, however, did not uncover any significant mutations. Subsequently, he began to experience walking difficulties, frequent falls, and trouble climbing stairs. At 6 years of age, he was presented to our hospital. His academic performance at school was deemed average. Upon physical examination, hypertrophy was noted in the calf and forearm muscles, along with weakness in the pelvic-girdle muscles. He also exhibited a positive Gowers’ sign and a wadding gait. A repeat serum creatine kinase (CK) assay revealed an elevated level of 6981U/L (normal: ≤190U/L). MRI scans of the bilateral hips and thigh muscles exhibited widespread long T2 signals and signs of myofascial edema. Meanwhile, the electrocardiogram showed a normal sinus rhythm, and echocardiography did not detect any cardiac abnormalities. The Wechsler Child Intelligence Scale assessed his overall IQ at 90. The clinical findings guided us toward a tentative diagnosis of dystrophinopathy. The patient underwent standard steroid treatment (prednisone 0,75mg/kg/d) and received regular followed-up. To confirm this diagnosis, we recommended the patient for LRS as a next step in our investigation. LRS revealed a 6.31Mb paracentric inversion between chromosomal regions chrXp21.3 and chrXp21.1. The inversion breakpoints were accurately mapped to genomic coordinates chrX:26,046,702 and chrX:32,353,866. Specifically, the breakpoint at chrX:26,046,702 lies within the intergenic space flanked by the RANBP1BP1 and MAGEB18 genes, whereas the breakpoint at chrX:32,353,866 falls within intron 41 of the DMD gene. In addition, the sequencing detected a 1bp deletion at chrX:26,046,701 and a 37bp deletion across chrX:32,353,828 − 32,353,865 in the patient’s genome (Fig. 5 ). The patient’s mother also found to carry this inversion in one of her X chromosomes. PCR/Sanger sequencing was performed to confirm the results obtained from LRS. Primers were specifically designed based on the LRS data (Fig. 2 ). Sanger sequencing accurately identified the inversion breakpoints at chrX:26,046,700/chrX:32,353,827 and chrX:26,046,702/chrX:32,353,866, signifying an inversion within these genomic coordinates on the X chromosome. Additionally, two deletions at chrX:26,046,701 and chrX:32,353,828 − 32,353,865 were confirmed in the patient’s DNA (Fig. 6 ). An indepth analysis of the breakpoints revealed the existence of simple repeats, denoted as (CATATA)n, at chrX:32,353,741 − 32,353,885. These simple repetitive sequences may lead to duplicate alignments during the genomic alignment process. Discussion and conclusions Substantial inversions affecting the DMD gene are an uncommon cause of DMD, with intrachromosomal inversions being exceptionally rare. To date, there have been 14 reported cases of X-chromosomal inversions that disrupt the DMD gene [ 2 , 8 – 18 ]. Our research has uncovered two previously undocumented intrachromosomal inversion variants disrupting the DMD gene in patients. The first case concerns a DMD patient with intellectual disability, who was found to have an approximately 110Mb pericentric inversion on the X chromosome. The second case involves another DMD patient, identified with an approximately 6.31Mb paracentric inversion on the X chromosome. Traditional diagnostic approaches like MLPA and WES were unsuccessful in identifying the mutations, whereas LRS proved to be efficacious and was instrumental in the diagnostic process. To date, intrachromosomal inversions associated with intellectual disability have been reported in 8 patients with DMD [ 11 – 13 , 15 – 18 ]. In 5 of these instances, an additional gene was implicated at the second breakpoint, potentially elucidating the cause of cognitive impairment [ 11 , 13 , 15 , 18 ]. In the first case we have presented, there are no known genes identified at the location of the second breakpoint. Previous researches suggest that cognitive and behavioral challenges are present in individuals with DMD and BMD. Approximately 30% of those with DMD have been reported to exhibit cognitive impairments, and around 40% face difficulties with reading [ 12 , 19 ]. Certain reports have suggested that in some patients with DMD cognitive impairment is linked to mutations that disrupt the production of the distal Dp140 and Dp71 dystrophin isoforms [ 20 – 22 ]. The dystrophin isoforms Dp71 and Dp140 are highly expressed in the brain, prompting the theory that they could be particularly crucial to the cognitive deficits observed in DMD. Dp71 synthesis begins between exons 62 and 63, whereas Dp140 starts upstream of exon 45, with its initial methionine codon situated in exon 51 [ 23 ]. In the first case from our study, the breakpoint located at chrX:31,927,491 falls within intron 47 of the DMD gene, disrupting its sequence. This disruption may impact the Dp140 isoform, potentially leading to compromised dystrophin-associated brain function. Nonetheless, the possibility of other contributing factors has not been ruled out. Repetitive DNA sequences play pivotal roles in generating genetic variation [ 24 ]. A variety of repetitive sequences have been identified as drivers for aberrant recombination events within the genome, which result in the formation of structural variants, including inversions [ 25 , 26 ]. Traditionally, repeated DNA sequences have been categorized as either interspersed or tandem repeats, distinguished by their respective positions and the mechanisms driving their expansion [ 27 ]. In previous reports concerning intrachromosomal inversions in patients with DMD, the breakpoints were predominantly located within interspersed repeats [ 2 , 12 , 14 ]. Simple repeated sequences (SRS) are defined as tandem repeats of microsatellite-sized (≤ 9bp units), minisatellite-sized (10-60bp units), or satellite-sized (> 60bp units) DNA sequences [ 27 ]. In the first case, (TA)n repeat sequences are located at the breakpoint, while in the second case, (CATATA)n repeat sequences are present at the breakpoint. Both (TA)n repeat sequences and (CATATA)n repeat sequences belong to microsatellite-sized SRS. To the best of our understanding, this represents the inaugural documentation of intrachromosomal inversions involving SRS in patients with DMD. SRS exhibit remarkable instability in terms of length, sequence composition, and copy number, with mutation rates typically 10–100,000 times higher than in other parts of the genome [ 28 ]. SRS might also play a role in regulating gene expression. This regulation could occur through the presence of binding sites for regulatory factors within these sequences or act as susceptible targets for epigenetic modifications [ 24 ]. Although muscle biopsy and RNA/cDNA sequencing are recommended by current guidelines for patients without detectable mutations using standard genetic testing methods such as MLPA or NGS [ 4 ], the invasive and painful nature of biopsies frequently results in patient apprehension, especially among children. LRS may offer a less invasive, time-efficient and effective alternative for the identification of rare mutations, including inversions. The identification of SRS at breakpoints in DMD patients assists in acquiring a more profound understanding of the mechanisms involved in structural variations, thereby facilitating exploration into potential treatments. Abbreviations DMD Duchenne Muscular Dystrophy MLPA Multiplex ligation probe amplification WES Whole exome sequencing NGS Next-generation sequencing LRS Long-read sequencing BMD Becker Muscular Dystrophy SNVs Single nucleotide variants SMART Single-molecule real-time sequencing IQ Intelligence quotient CK Creatine kinase SRS Simple repeated sequences Declarations Acknowledgements We would like to thank the patients and their parents for their support of our research. Author’s contributions LC drafted the manuscript and prepared the figures. YL did the follow-up with the patient. XL and YL edited the manuscript. YT and HW edited the figures. All authors contributed to the article and approved the submitted version. Funding This work was supported by grants from the Natural Science Foundation of Hubei Province Project (2022CFB203). Availability of data and materials The raw datasets generated and analyzed during the current study are not publicly available in order to protect participant confidentiality. The datasets obtained during the current study are available from the corresponding author if the requirements are reasonable. Ethics approval and consent to participate This study was approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. All study procedures were conducted in accordance with the tenets of the Declaration of Helsinki. The patient and his parents provided written informed consent to participate in this study. Consent for publication Written informed consent to publish this case was obtained from the patients and his parents, including case description and medical data. Completing interests All authors declare that they have no conflict of interest. References Kunkel LM, Monaco AP, Middlesworth W, et al . Specific cloning of DNA fragments absent from the DNA of a male patient with an X chromosome deletion. Proc Natl Acad Sci USA . 1985; 82:4778–4782 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 . 2023;31(5):504-511. Muntoni F, Torelli S, Ferlini A. Dystrophin and mutations: one gene, several proteins, multiple phenotypes. Lancet Neurol . 2003; 2:731–740 Fratter C, Dalgleish R, Allen SK, et al . EMQN best practice guidelines for genetic testing in dystrophinopathies. Eur J Hum Genet . 2020; 28:1141–59. Kong XD, Zhong XJ, Liu LN, et al . 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The Xq22 inversion breakpoint interrupted a novel Ras-like GTPase gene in a patient with Duchenne muscular dystrophy and profound mental retardation. Am J Hum Genet . 2002;71(3):637-645. Folland C, Ganesh V, Weisburd B, et al . Transcriptome and Genome Analysis Uncovers a DMD Structural Variant: A Case Report. Neurol Genet . 2023;9(2): e200064. Erbe LS, Hoffjan S, Janßen S, et al . Exome Sequencing and Optical Genome Mapping in Molecularly Unsolved Cases of Duchenne Muscular Dystrophy: Identification of a Causative X-Chromosomal Inversion Disrupting the DMD Gene. Int J Mol Sci . 2023;24(19):14716. Chandrasekhar A, Mroczkowski HJ, Urraca N, et al . Genome sequencing detects a balanced pericentric inversion with breakpoints that impact the DMD and upstream region of POU3F4 gene. Am J Med Genet A . 2023;10.1002 Cotton S, Voudouris NJ, Greenwood KM. Intelligence and Duchenne muscular dystrophy: full-scale, verbal, and performance intelligence quotients. Dev Med Child Neurol . 2001;43(7):497-501. Taylor PJ, Betts GA, Maroulis S, et al . Dystrophin gene mutation location and the risk of cognitive impairment in Duchenne muscular dystrophy. PLoS One . 2010;5(1): e8803 Giliberto F, Ferreiro V, Dalamon V, et al . Dystrophin deletions and cognitive impairment in Duchenne/Becker muscular dystrophy. Neurol Res . 2004, 26:83-87 Bovolenta M, Neri M, Fini S, et al . A novel custom high density-comparative genomic hybridization array detects common rearrangements as well as deep intronic mutations in dystrophinopathies. BMC Genomics . 2008; 9:572. Moizard MP, Billard C, Toutain A, et al . Are Dp71 and Dp140 brain dystrophin isoforms related to cognitive impairment in Duchenne muscular dystrophy?. Am J Med Genet . 1998;80(1):32-41. Liao X, Zhu W, Zhou J, et al . Repetitive DNA sequence detection and its role in the human genome. Communications Biology . 2023,6:954. Weckselblatt B, Rudd MK. Human structural variation: mechanisms of chromosome rearrangements. Trends Genet . 2015; 31:587–589. Carvalho CM, Ramocki MB, Pehlivan D, et al . Inverted genomic segments and complex triplication rearrangements are mediated by inverted repeats in the human genome. Nat Genet . 2011; 43:1074. Chung TH, Zhuravskaya A, Makeyev EV. Regulation potential of transcribed simple repeated sequences in developing neurons. Hum. Genet . Published online December 28, 2023. Gymrek M, Willems T, Reich D, et al . Interpreting short tandem repeat variations in humans using mutational constraint. Nat. Genet . 2017, 49: 1495–1501. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 09 Sep, 2024 Read the published version in BMC Medical Genomics → Version 1 posted Editorial decision: Revision requested 27 Mar, 2024 Submission checks completed at journal 20 Mar, 2024 Editor assigned by journal 20 Mar, 2024 First submitted to journal 23 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3982190","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":281796045,"identity":"299a3d68-6d06-4d46-997f-4ea28b4f8a09","order_by":0,"name":"Liqing Chen","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Liqing","middleName":"","lastName":"Chen","suffix":""},{"id":281796046,"identity":"cc42d563-1d22-455d-b504-a4de2572aa8e","order_by":1,"name":"Xiaoping Luo","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Xiaoping","middleName":"","lastName":"Luo","suffix":""},{"id":281796049,"identity":"59e7c327-f7c9-4cc4-a28f-95c5d41f1b5b","order_by":2,"name":"Hongling Wang","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Hongling","middleName":"","lastName":"Wang","suffix":""},{"id":281796050,"identity":"015d9ddd-b754-4cef-9a43-64c1765557c5","order_by":3,"name":"Yu Tian","email":"","orcid":"","institution":"Grandomics (China)","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Tian","suffix":""},{"id":281796051,"identity":"691b50d8-72d6-42eb-9acd-4e911bd54de9","order_by":4,"name":"Yan Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYBACPiBm/GBgI8fPzHz4AVFa2ICYWaIizViynS3NgGgtDDxnDiduOM+jIEGcFonkZw8k25gZNx/mYTBgqLGJJkJLmrlBYRsbs9lh3gMPGI6l5TYQ1pJgJiHZxsNmdpgvwYCx4TAxWtK/SfC2SfAYN/MYSBCpJcdMgueMgYQBM9FaeN6USUtUJBhIHAYGcgIxfuFnT98m+cHgf31//+HDDz7U2BDWwiCQgMRJwKEIzZoDRCkbBaNgFIyCkQwA75Y3icWUIhIAAAAASUVORK5CYII=","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Yan","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-02-23 14:48:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3982190/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3982190/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12920-024-01997-2","type":"published","date":"2024-09-09T15:57:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53256194,"identity":"7f8984b7-f3e2-451a-897c-ed3b1af3d901","added_by":"auto","created_at":"2024-03-22 13:31:40","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":83322,"visible":true,"origin":"","legend":"\u003cp\u003ePrimers in the Sanger sequencing\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3982190/v1/2ed20d0141fdcbbd89edd123.jpg"},{"id":53256198,"identity":"1eb0944a-f7bf-4fc8-bf41-e18ad9026d08","added_by":"auto","created_at":"2024-03-22 13:31:40","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":110768,"visible":true,"origin":"","legend":"\u003cp\u003eThe long-read sequencing revealed an inversion variant in patient 1.\u003c/p\u003e\n\u003cp\u003eThis inversion spans a vast distance of 110.81Mb, stretching from chrX:31927491 (Xp21.1) to chrX:142742006 (Xq27.3), crossing the centromere on the X chromosome. Additionally, a 9bp deletion spanning chrX:31,927,482-31,927,490 and a 313bp duplication within chrX:142,741,694-142,742,006 in the patient’s DNA were identified.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3982190/v1/8da9c3d3b64cfb7fbe8b2d97.jpg"},{"id":53256195,"identity":"bf1609ac-6913-457f-a120-762b50bedfed","added_by":"auto","created_at":"2024-03-22 13:31:40","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":105718,"visible":true,"origin":"","legend":"\u003cp\u003eDiagrammatic representation of the gene rearrangement in the patient\u003c/p\u003e\n\u003cp\u003eA: Patient’s gene showing the inverted and duplicated segments and mutation breakpoints.\u003c/p\u003e\n\u003cp\u003eB: The proximal mutation breakpoint sequence\u003c/p\u003e\n\u003cp\u003eC: The distal mutation breakpoint sequence\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3982190/v1/18ea80d5b797c0d9c4b2ada5.jpg"},{"id":53256193,"identity":"fc74884a-c89d-47c0-a651-d94dda9195fb","added_by":"auto","created_at":"2024-03-22 13:31:40","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":25742,"visible":true,"origin":"","legend":"\u003cp\u003eThe family pedigree of the patient.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3982190/v1/2d7e11bf7fa92ac644ceae2c.jpg"},{"id":53256197,"identity":"068c753c-dfc7-4105-9a74-014f52d8d9fe","added_by":"auto","created_at":"2024-03-22 13:31:40","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":85760,"visible":true,"origin":"","legend":"\u003cp\u003eThe long-read sequencing revealed an inversion variant in patient 2.\u003c/p\u003e\n\u003cp\u003eThis paracentric inversion spans a distance of 6.31Mb, stretching from chrX:26046702 (Xp21.3) to chrX:32353866 (Xp21.1). Additionally, a 1bp deletion on the chrX:26,046,701 and a 37bp deletion spanning chrX:32,353,828-32,353,865 in the patient’s DNA were identified.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3982190/v1/4a5fe94951de6dadc865fcf7.jpg"},{"id":53256199,"identity":"a7456cb3-dc65-42a8-84f1-2b59777e5980","added_by":"auto","created_at":"2024-03-22 13:31:41","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":64496,"visible":true,"origin":"","legend":"\u003cp\u003eDiagrammatic representation of the gene rearrangement in the patient\u003c/p\u003e\n\u003cp\u003eA: Patient’s gene showing the inverted segments and mutation breakpoints.\u003c/p\u003e\n\u003cp\u003eB: The sanger sequence of the proximal mutation breakpoint sequence of the patient 2\u003c/p\u003e\n\u003cp\u003eC: The sanger sequence of the distal mutation breakpoint sequence of the patient 2\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3982190/v1/7e5fac0d4a5827dbdfd23a53.jpg"},{"id":64619111,"identity":"f61dcc39-eef3-4132-a1f6-cb473398ba7a","added_by":"auto","created_at":"2024-09-16 16:11:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":822267,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3982190/v1/48cc1679-e265-4d27-81eb-f908b9db0947.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identifying inversions with Breakpoints in the Dystrophin Gene through Long-Read Sequencing: Report of Two Cases","fulltext":[{"header":"Background","content":"\u003cp\u003eThe dystrophin gene (\u003cem\u003eDMD\u003c/em\u003e; MIM #300377), situated within a 2.4 Mb region on the X chromosome at the Xp21.2 locus, demonstrates complexity by producing multiple isoforms across a range of tissues. Mutations in this gene can lead to the absence of functional dystrophin protein, primarily causing progressive degradation of muscle tissue, including skeletal, myocardial, and smooth muscle. This also can result in orthopedic and respiratory complications [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Loss-of-function mutations in the dystrophin gene are responsible for Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD) and X-linked dilated cardiomyopathy. These mutations cover a wide spectrum of alterations, including deletions, duplications, single nucleotide variants (SNVs), insertions and complex rearrangements [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Among them, intragenic deletions affecting one or more exons are the most common, making up roughly 60% of all dystrophin mutations, with duplications being less frequent, appearing at a rate of 6 to 8% [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Complex rearrangements, like balanced translocations or inversions, are exceptionally uncommon.\u003c/p\u003e \u003cp\u003eIn investigating causative mutations in DMD/BMD patients, various methods have been utilized. Multiplex ligation-dependent probe amplification (MLPA) is the conventional method used in genetic testing to detect exonic deletions and duplications. In case where this analysis does not reveal a pathogenic mutation, the next step is to perform whole exome sequencing (WES) using next-generation sequencing (NGS) to analyze the complete coding region of the DMD gene [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The diagnostic rate can exceed 95% [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The undetected mutations that remain are primarily deep intronic mutations [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These can be identified through RNA analysis of muscle tissue using RT-PCR or short-read sequencing. Nonetheless, a subset of patients remains undiagnosed genetically, even with the application of these techniques, owing to the existence of exceptionally rare and complex structural variants.\u003c/p\u003e \u003cp\u003eA previous study demonstrated that the captured long-read sequencing (LRS) panel effectively detected a spectrum of DMD mutations, ranging from SNVs to structural variations [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. LRS, exemplified by the Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PB) single-molecule real-time sequencing (SMRT) platforms, facilitated a more precise identification of breakpoint positions for structural variations in the DMD gene. The precision in identifying breakpoints enables the decoding of the entire sequence within the DMD rearrangement region. Because the errors in the sequencing data obtained through PB sequencing were more consistent or aligned with each other, making PB easier to identify the correct sequence than ONT. In this study, we identified two intrachromosomal inversions involving the dystrophin gene within two unrelated families using LRS facilitated by the PB SMRT platform. In both cases, simple repeat sequences were observed at the breakpoints of these inversions.\u003c/p\u003e"},{"header":"Case presentation","content":"\u003cp\u003ePatient 1\u003c/p\u003e \u003cp\u003eA ten-year-old boy was admitted to our hospital due to muscle weakness. He is the elder of two brothers born to healthy, non-consanguineous Chinese parents with no significant personal or family medical history. The patient achieved independent walking at the age of fourteen months. At the age of five, he began to experience a gradual decline in his walking abilities, becoming prone to falls. Currently, he experiences difficulties in jumping and getting up after squatting. Moreover, he exhibits with intellectual disability and faces challenges in his learning at school, leading to lower grades in his examinations. His physical examination revealed hypertrophy of the gastrocnemius muscle, a positive Gowers\u0026rsquo; sign and a waddling gait. His head circumference measured 50cm and his tendon reflexes showed weakness. Based on the provided information, we initially diagnosed dystrophinopathy clinically. A serum creatine kinase test was conducted, revealing a significantly elevated level of 10961U/L (normal\u0026thinsp;\u0026le;\u0026thinsp;190U/L). The Wechsler Child Intelligence Scale assessed his overall IQ at 65. The electrocardiography results fell within the normal range, but the color Doppler echocardiography examination indicated left ventricular enlargement.\u003c/p\u003e \u003cp\u003eFollowing the clinical diagnosis of dystrophinopathy, the patient underwent standard steroid treatment (prednisone 0,75mg/kg/d) and received regular followed-up. Genetic tests were initiated subsequently. Initially, we conducted MLPA, which did not indicate any deletions or duplication. Subsequently, we performed targeted WES using NGS to identify SNVs or splicing mutations across all 79 exons of the \u003cem\u003eDMD\u003c/em\u003e gene. Unfortunately, no causative genetic mutations were identified through these approaches. Based on previous reports, the patient then underwent LRS directly [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA significant pericentric inversion, spanning 110.81Mb and mapping between chrXp21.1 and chrXq27.3, was identified through LRS. The inversion breakpoints were precisely located at the genomic positions of chrX:31,927,491 and chrX:142,742,006. Notably, the breakpoint at chrX:31,927,491 is situated within intron 47 of the \u003cem\u003eDMD\u003c/em\u003e gene. Furthermore, the patient\u0026rsquo;s DNA revealed a 9bp deletion spanning chrX:31,927,482\u0026thinsp;\u0026minus;\u0026thinsp;31,927,490 (CTTTGGGAA) and a 313bp duplication within chrX:142,741,694\u0026thinsp;\u0026minus;\u0026thinsp;142,742,006 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe mutation identified through LRS was subsequently confirmed by PCR/Sanger sequencing. PCR/Sanger sequencing is a widely used sequencing technology employed to validate and accurately determine the breakpoint location in inversion mutations. Two primers were designed based on the LRS results, and their specific details can be found in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The inversion breakpoints were accurately determined through Sanger sequencing to be at chrX:31,927,481/142,742,828 and chrX:31,927,491/142,741,748, denoting an inversion between these genomic coordinates on the X chromosome. Additionally, a 9bp deletion at chrX:31,927,482\u0026thinsp;\u0026minus;\u0026thinsp;31,927,490 and an 81bp duplication at chrX:142,741,748\u0026thinsp;\u0026minus;\u0026thinsp;142,741,828 were identified at these breakpoints, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Detailed analysis of the breakpoints uncovered the presence of LTR sequences at chrX:31,927,411\u0026thinsp;\u0026minus;\u0026thinsp;31,927,629 (219bp) and (TA)n sequences at chrX:142,741,289\u0026thinsp;\u0026minus;\u0026thinsp;142,741,461 (173bp), chrX142,741,503\u0026thinsp;\u0026minus;\u0026thinsp;142,741,649 (147bp), chrX142,741,665\u0026thinsp;\u0026minus;\u0026thinsp;142,741,824 (160bp) and chrX:142,741,855\u0026thinsp;\u0026minus;\u0026thinsp;142,742,166 (312bp) respectively. The presence of these simple repetitive sequences surrounds both breakpoints, resulting in duplicate alignments during the alignment process. Consequently, this can lead to differences between the results of long-read sequencing and Sanger sequencing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePatient 2\u003c/p\u003e \u003cp\u003eThe second case concerns a 6-year-old boy who was admitted to our hospital presenting with abnormal gait. A confirmed family history revealed that similar conditions had afflicted his maternal uncle and cousin, both of whom exhibited comparable symptoms and sadly succumbed to their illness after reaching the age of 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The boy took his first steps at 18 months. At the age of 3, he was hospitalized for a fever at a different institution, where tests revealed an abnormally high serum creatine kinase level (CK\u0026thinsp;=\u0026thinsp;17215U/L, normal range: 24-204U/L). Despite these findings, he did not display any substantial motor deficits at that juncture. To establish a definitive diagnosis, he was subjected to MLPA and WES to identify common deletions and duplications within the DMD gene. These tests, however, did not uncover any significant mutations. Subsequently, he began to experience walking difficulties, frequent falls, and trouble climbing stairs. At 6 years of age, he was presented to our hospital. His academic performance at school was deemed average. Upon physical examination, hypertrophy was noted in the calf and forearm muscles, along with weakness in the pelvic-girdle muscles. He also exhibited a positive Gowers\u0026rsquo; sign and a wadding gait. A repeat serum creatine kinase (CK) assay revealed an elevated level of 6981U/L (normal: \u0026le;190U/L). MRI scans of the bilateral hips and thigh muscles exhibited widespread long T2 signals and signs of myofascial edema. Meanwhile, the electrocardiogram showed a normal sinus rhythm, and echocardiography did not detect any cardiac abnormalities. The Wechsler Child Intelligence Scale assessed his overall IQ at 90. The clinical findings guided us toward a tentative diagnosis of dystrophinopathy. The patient underwent standard steroid treatment (prednisone 0,75mg/kg/d) and received regular followed-up. To confirm this diagnosis, we recommended the patient for LRS as a next step in our investigation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLRS revealed a 6.31Mb paracentric inversion between chromosomal regions chrXp21.3 and chrXp21.1. The inversion breakpoints were accurately mapped to genomic coordinates chrX:26,046,702 and chrX:32,353,866. Specifically, the breakpoint at chrX:26,046,702 lies within the intergenic space flanked by the RANBP1BP1 and MAGEB18 genes, whereas the breakpoint at chrX:32,353,866 falls within intron 41 of the \u003cem\u003eDMD\u003c/em\u003e gene. In addition, the sequencing detected a 1bp deletion at chrX:26,046,701 and a 37bp deletion across chrX:32,353,828\u0026thinsp;\u0026minus;\u0026thinsp;32,353,865 in the patient\u0026rsquo;s genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The patient\u0026rsquo;s mother also found to carry this inversion in one of her X chromosomes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePCR/Sanger sequencing was performed to confirm the results obtained from LRS. Primers were specifically designed based on the LRS data (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Sanger sequencing accurately identified the inversion breakpoints at chrX:26,046,700/chrX:32,353,827 and chrX:26,046,702/chrX:32,353,866, signifying an inversion within these genomic coordinates on the X chromosome. Additionally, two deletions at chrX:26,046,701 and chrX:32,353,828\u0026thinsp;\u0026minus;\u0026thinsp;32,353,865 were confirmed in the patient\u0026rsquo;s DNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). An indepth analysis of the breakpoints revealed the existence of simple repeats, denoted as (CATATA)n, at chrX:32,353,741\u0026thinsp;\u0026minus;\u0026thinsp;32,353,885. These simple repetitive sequences may lead to duplicate alignments during the genomic alignment process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion and conclusions","content":"\u003cp\u003eSubstantial inversions affecting the \u003cem\u003eDMD\u003c/em\u003e gene are an uncommon cause of DMD, with intrachromosomal inversions being exceptionally rare. To date, there have been 14 reported cases of X-chromosomal inversions that disrupt the \u003cem\u003eDMD\u003c/em\u003e gene [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12 CR13 CR14 CR15 CR16 CR17\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Our research has uncovered two previously undocumented intrachromosomal inversion variants disrupting the \u003cem\u003eDMD\u003c/em\u003e gene in patients. The first case concerns a DMD patient with intellectual disability, who was found to have an approximately 110Mb pericentric inversion on the X chromosome. The second case involves another DMD patient, identified with an approximately 6.31Mb paracentric inversion on the X chromosome. Traditional diagnostic approaches like MLPA and WES were unsuccessful in identifying the mutations, whereas LRS proved to be efficacious and was instrumental in the diagnostic process.\u003c/p\u003e \u003cp\u003eTo date, intrachromosomal inversions associated with intellectual disability have been reported in 8 patients with DMD [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In 5 of these instances, an additional gene was implicated at the second breakpoint, potentially elucidating the cause of cognitive impairment [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In the first case we have presented, there are no known genes identified at the location of the second breakpoint. Previous researches suggest that cognitive and behavioral challenges are present in individuals with DMD and BMD. Approximately 30% of those with DMD have been reported to exhibit cognitive impairments, and around 40% face difficulties with reading [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Certain reports have suggested that in some patients with DMD cognitive impairment is linked to mutations that disrupt the production of the distal Dp140 and Dp71 dystrophin isoforms [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The dystrophin isoforms Dp71 and Dp140 are highly expressed in the brain, prompting the theory that they could be particularly crucial to the cognitive deficits observed in DMD. Dp71 synthesis begins between exons 62 and 63, whereas Dp140 starts upstream of exon 45, with its initial methionine codon situated in exon 51 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In the first case from our study, the breakpoint located at chrX:31,927,491 falls within intron 47 of the \u003cem\u003eDMD\u003c/em\u003e gene, disrupting its sequence. This disruption may impact the Dp140 isoform, potentially leading to compromised dystrophin-associated brain function. Nonetheless, the possibility of other contributing factors has not been ruled out.\u003c/p\u003e \u003cp\u003eRepetitive DNA sequences play pivotal roles in generating genetic variation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. A variety of repetitive sequences have been identified as drivers for aberrant recombination events within the genome, which result in the formation of structural variants, including inversions [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Traditionally, repeated DNA sequences have been categorized as either interspersed or tandem repeats, distinguished by their respective positions and the mechanisms driving their expansion [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In previous reports concerning intrachromosomal inversions in patients with DMD, the breakpoints were predominantly located within interspersed repeats [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Simple repeated sequences (SRS) are defined as tandem repeats of microsatellite-sized (\u0026le;\u0026thinsp;9bp units), minisatellite-sized (10-60bp units), or satellite-sized (\u0026gt;\u0026thinsp;60bp units) DNA sequences [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In the first case, (TA)n repeat sequences are located at the breakpoint, while in the second case, (CATATA)n repeat sequences are present at the breakpoint. Both (TA)n repeat sequences and (CATATA)n repeat sequences belong to microsatellite-sized SRS. To the best of our understanding, this represents the inaugural documentation of intrachromosomal inversions involving SRS in patients with DMD. SRS exhibit remarkable instability in terms of length, sequence composition, and copy number, with mutation rates typically 10\u0026ndash;100,000 times higher than in other parts of the genome [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. SRS might also play a role in regulating gene expression. This regulation could occur through the presence of binding sites for regulatory factors within these sequences or act as susceptible targets for epigenetic modifications [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough muscle biopsy and RNA/cDNA sequencing are recommended by current guidelines for patients without detectable mutations using standard genetic testing methods such as MLPA or NGS [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], the invasive and painful nature of biopsies frequently results in patient apprehension, especially among children. LRS may offer a less invasive, time-efficient and effective alternative for the identification of rare mutations, including inversions. The identification of SRS at breakpoints in DMD patients assists in acquiring a more profound understanding of the mechanisms involved in structural variations, thereby facilitating exploration into potential treatments.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eDMD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Duchenne Muscular Dystrophy\u003c/p\u003e\n\u003cp\u003eMLPA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Multiplex ligation probe amplification\u003c/p\u003e\n\u003cp\u003eWES \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Whole exome sequencing\u003c/p\u003e\n\u003cp\u003eNGS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Next-generation sequencing\u003c/p\u003e\n\u003cp\u003eLRS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Long-read sequencing\u003c/p\u003e\n\u003cp\u003eBMD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Becker Muscular Dystrophy\u003c/p\u003e\n\u003cp\u003eSNVs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Single nucleotide variants\u003c/p\u003e\n\u003cp\u003eSMART \u0026nbsp; \u0026nbsp; \u0026nbsp; Single-molecule real-time sequencing\u003c/p\u003e\n\u003cp\u003eIQ \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Intelligence quotient\u003c/p\u003e\n\u003cp\u003eCK \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Creatine kinase\u003c/p\u003e\n\u003cp\u003eSRS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Simple repeated sequences\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the patients and their parents for their support of our research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLC drafted the manuscript and prepared the figures. YL did the follow-up with the patient. XL and YL edited the manuscript. YT and HW edited the figures. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the Natural Science Foundation of Hubei Province Project (2022CFB203).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw datasets generated and analyzed during the current study are not publicly available in order to protect participant confidentiality. The datasets obtained during the current study are available from the corresponding author if the requirements are reasonable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. All study procedures were conducted in accordance with the tenets of the Declaration of Helsinki. The patient and his parents provided written informed consent to participate in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent to publish this case was obtained from the patients and his parents, including case description and medical data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompleting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKunkel LM, Monaco AP, Middlesworth W, \u003cem\u003eet al\u003c/em\u003e. Specific cloning of DNA fragments absent from the DNA of a male patient with an X chromosome deletion. \u003cem\u003eProc Natl Acad Sci USA\u003c/em\u003e. 1985; 82:4778\u0026ndash;4782\u003c/li\u003e\n\u003cli\u003eGeng C, Zhang C, Li P, \u003cem\u003eet al\u003c/em\u003e. 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Molecular characterization of an X (p21.2; q28) chromosomal inversion in a Duchenne muscular dystrophy patient with mental retardation reveals a novel long non-coding gene on Xq28. \u003cem\u003eJ Hum Genet\u003c/em\u003e. 2013;58(1):33-39.\u003c/li\u003e\n\u003cli\u003eFalzarano MS, Grilli A, Zia S, \u003cem\u003eet al\u003c/em\u003e. RNA-seq in DMD urinary stem cells recognized muscle-related transcription signatures and addressed the identification of atypical mutations by whole-genome sequencing. \u003cem\u003eHGG Adv\u003c/em\u003e. 2021;3(1):100054.\u003c/li\u003e\n\u003cli\u003eSaito-Ohara F, Fukuda Y, Ito M, \u003cem\u003eet al\u003c/em\u003e. The Xq22 inversion breakpoint interrupted a novel Ras-like GTPase gene in a patient with Duchenne muscular dystrophy and profound mental retardation. \u003cem\u003eAm J Hum Genet\u003c/em\u003e. 2002;71(3):637-645. \u003c/li\u003e\n\u003cli\u003eFolland C, Ganesh V, Weisburd B, \u003cem\u003eet al\u003c/em\u003e. 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Intelligence and Duchenne muscular dystrophy: full-scale, verbal, and performance intelligence quotients. \u003cem\u003eDev Med Child Neurol\u003c/em\u003e. 2001;43(7):497-501. \u003c/li\u003e\n\u003cli\u003eTaylor PJ, Betts GA, Maroulis S, \u003cem\u003eet al\u003c/em\u003e. Dystrophin gene mutation location and the risk of cognitive impairment in Duchenne muscular dystrophy. \u003cem\u003ePLoS One\u003c/em\u003e. 2010;5(1): e8803\u003c/li\u003e\n\u003cli\u003eGiliberto F, Ferreiro V, Dalamon V, \u003cem\u003eet al\u003c/em\u003e. Dystrophin deletions and cognitive impairment in Duchenne/Becker muscular dystrophy. \u003cem\u003eNeurol Res\u003c/em\u003e. 2004, 26:83-87\u003c/li\u003e\n\u003cli\u003eBovolenta M, Neri M, Fini S, \u003cem\u003eet al\u003c/em\u003e. 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Human structural variation: mechanisms of chromosome rearrangements. \u003cem\u003eTrends Genet\u003c/em\u003e. 2015; 31:587\u0026ndash;589.\u003c/li\u003e\n\u003cli\u003eCarvalho CM, Ramocki MB, Pehlivan D, \u003cem\u003eet al\u003c/em\u003e. Inverted genomic segments and complex triplication rearrangements are mediated by inverted repeats in the human genome. \u003cem\u003eNat Genet\u003c/em\u003e. 2011; 43:1074.\u003c/li\u003e\n\u003cli\u003eChung TH, Zhuravskaya A, Makeyev EV. Regulation potential of transcribed simple repeated sequences in developing neurons. \u003cem\u003eHum. Genet\u003c/em\u003e. Published online December 28, 2023.\u003c/li\u003e\n\u003cli\u003eGymrek M, Willems T, Reich D, \u003cem\u003eet al\u003c/em\u003e. Interpreting short tandem repeat variations in humans using mutational constraint. \u003cem\u003eNat. Genet\u003c/em\u003e. 2017, 49: 1495\u0026ndash;1501.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-medical-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mgnm","sideBox":"Learn more about [BMC Medical Genomics](http://bmcmedgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mgnm/default.aspx","title":"BMC Medical Genomics","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"DMD, inversion, breakpoint, long-read sequencing, simple repeated sequences","lastPublishedDoi":"10.21203/rs.3.rs-3982190/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3982190/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eDuchenne Muscular Dystrophy (DMD) is an X-linked disorder caused by mutations in the \u003cem\u003eDMD\u003c/em\u003e gene, with large deletions being the most frequent type of mutation. Large inversions involving the \u003cem\u003eDMD\u003c/em\u003e gene are a less common cause of the disorder, primarily because they often elude detection by standard diagnostic methods such as multiplex ligation probe amplification (MLPA) and whole exome sequencing (WES) utilizing next-generation sequencing (NGS) technologies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCase presentation\u003c/strong\u003e: Our research uncovered two intrachromosomal inversions involved the dystrophin gene in two unrelated families through Long-read sequencing (LRS). To confirm these variants, Sanger sequencing subsequently carried out. The first case involved a pericentric inversion from \u003cem\u003eDMD\u003c/em\u003e intron 47 to the Xq27.3. The second case featured a paracentric inversion between \u003cem\u003eDMD\u003c/em\u003e intron 42 and Xp21.1, inherited from the mother. In both cases, simple repeat sequences (SRS) were present at the breakpoints of these inversions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: Our findings demonstrate that LRS can be effectively used to detect atypical mutation. The identification of SRS at breakpoints in DMD patients assists in acquiring a more profound understanding of the mechanisms involved in structural variations, thereby facilitating exploration into potential treatments.\u003c/p\u003e","manuscriptTitle":"Identifying inversions with Breakpoints in the Dystrophin Gene through Long-Read Sequencing: Report of Two Cases","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-22 13:31:35","doi":"10.21203/rs.3.rs-3982190/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-27T05:56:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-20T11:42:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-20T11:42:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Medical Genomics","date":"2024-02-23T14:40:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-medical-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mgnm","sideBox":"Learn more about [BMC Medical Genomics](http://bmcmedgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mgnm/default.aspx","title":"BMC Medical Genomics","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c7f6cbfd-a8e8-4f06-9799-7c6b9c9917f5","owner":[],"postedDate":"March 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-16T16:02:14+00:00","versionOfRecord":{"articleIdentity":"rs-3982190","link":"https://doi.org/10.1186/s12920-024-01997-2","journal":{"identity":"bmc-medical-genomics","isVorOnly":false,"title":"BMC Medical Genomics"},"publishedOn":"2024-09-09 15:57:31","publishedOnDateReadable":"September 9th, 2024"},"versionCreatedAt":"2024-03-22 13:31:35","video":"","vorDoi":"10.1186/s12920-024-01997-2","vorDoiUrl":"https://doi.org/10.1186/s12920-024-01997-2","workflowStages":[]},"version":"v1","identity":"rs-3982190","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3982190","identity":"rs-3982190","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}