Adaptive long-read and transcriptome sequencing detail a submicroscopic inv(15)(q14q15), generating fusion transcripts and MEIS2 and NUSAP1 haploinsufficiency

Adaptive long-read and transcriptome sequencing detail a submicroscopic inv(15)(q14q15), generating fusion transcripts and MEIS2 and NUSAP1 haploinsufficiency | 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 Article Adaptive long-read and transcriptome sequencing detail a submicroscopic inv(15)(q14q15), generating fusion transcripts and MEIS2 and NUSAP1 haploinsufficiency Marlene Ek, Malin Kvarnung, Maria Pettersson, Maria Johansson Soller, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5112053/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Dec, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Inversions are balanced structural variants that often remain undetected in genetic diagnostics. We present a female proband with a de novo Chromosome 15 paracentric inversion, disrupting MEIS2 and NUSAP1 . The inversion was detected by short-read genome sequencing and confirmed with adaptive long-read sequencing. The breakpoint junction analysis revealed a 96 base pair (bp) deletion and an 18 bp insertion in the two junctions, suggesting that the rearrangement arose through a replicative error. Transcriptome sequencing of cultured fibroblasts revealed normal MEIS2 levels and 0.61-fold decreased expression of NUSAP1 . Furthermore, two fusion transcripts were detected and confirmed by Sanger sequencing. Heterozygous loss of MEIS2 (MIM# 600987) is associated with a cleft palate, heart malformations, and intellectual impairment, which overlap with the clinical symptoms observed in the proband. The observed fusion transcripts are likely non-functional, and MEIS2 haploinsufficiency is the likely disease causative mechanism. Altogether, this study findings illustrate the importance of including inversions in rare disease diagnostic testing and highlight the value of long read sequencing for the validation and characterization of such variants. Health sciences/Medical research Health sciences/Molecular medicine Health sciences/Neurology Health sciences/Diseases/Neurological disorders Inversion adaptive long-read genome sequencing RNA sequencing MEIS2 NUSAP1 gene disruption Figures Figure 1 Figure 2 INTRODUCTION Pathogenic variants in MEIS2 are associated with a syndrome known as “cleft palate, cardiac defects, and impaired intellectual development” (CPCMR, MIM# 600987). MEIS2 , a homeodomain-containing protein, is part of the three amino acid loop extension (TALE) family, and functions as a transcription regulator. Meis2 has been implicated in the development of limbs and brain in chicks, lens and retina formation in mice and Mekada fish, and is essential for both cardiac and neural crest development in mice. (1–5) The gene is highly intolerant to loss-of-function mutations, with a pLI score greater than 0.9, and causes disease primarily through haploinsufficiency. Most of the described MEIS2 variants are de novo , although cases of inherited variants have been described. Carrier parents tend to exhibit a similar phenotype, with mosaic carriers showing milder expression. (6–10) The previously reported disease-causing variants in MEIS2 include both structural variants and sequence variants, such as single nucleotide variants (SNVs) and insertions/deletions (INDELs). The structural variants are predominantly copy number variants (CNVs), with deletions, ranging in size from 0.6 to 6.97 Mb, and one family reported with a 58 kb tandem duplication. One of the deletions was part of a de novo complex chromosomal rearrangement. (6–8, 11–18) Notably, one case involving a balanced structural variant has been reported: a balanced reciprocal translocation between chromosomes 11 and 15, t(11;15)(p14;q14), where the breakpoint on chromosome 15 disrupts MEIS2 in intron 6. (19) The reported SNV/INDELs include truncating, splice site, missense variants or in-frame deletions within the homeodomain. (9, 10, 18, 20–27) We detail the genetic aberrations in a proband with cleft palate and high-functioning autism. Structural variant analysis of short-read genome sequencing revealed a 4.37 Mb inversion on chromosome 15, with breakpoints in intron 8 of MEIS2 and intron 7 of NUSAP1 , generating two fusion transcripts. RESULTS The proband is the first child of healthy, non-consanguineous parents. She was born after an uneventful pregnancy at 39 weeks + 6 days (birth weight 3670 g, birth length 53 cm and head circumference 35 cm). A posterior cleft palate was diagnosed early, and she experienced feeding difficulties during the first months of life. The cleft palate was surgically corrected twice, at 5 and 12 months of age. Her motor development was delayed; she sat unsupported at 9 months and walked independently at 19 months of age. She exhibited hypotonia and hypermobile joints, and her postnatal growth was slightly restricted, with a head circumference of -2 SD and a length of -0,5 SD at 3 years of age. Adhesions between labia majora initially raised suspicion of urethral duplication, but an ultrasound of the urinary tracts was normal. A cardiac ultrasound revealed a small atrial septal defect, which was clinically insignificant. A neuropsychological evaluation at the age of 5.5 years diagnosed her with high-function autism and her intellectual level was within the normal range. Some facial features were present, including penciled eyebrows, rounded eyes, large and protruding ears, and discrete fetal finger pads. She has two younger, healthy siblings. Genetic tests included array comparative genomic hybridization at 18 months of age, short-read genome sequencing analysis with in-silico gene panels for neuromuscular disorders (499 genes) and intellectual disability (885 genes) at age 7, all with normal results. Genome-wide structural variant analysis of the short-read genome sequencing data identified a 4.37 Mb inversion on chromosome 15, with breakpoint 1 (BP1), localized in intron 8 of the MANE transcript NM_170675.5 of MEIS2 and breakpoint 2 (BP2) in intron 7 of NUSAP1 (NM_016359.5). Follow-up analysis of the parents confirmed that the variant had arisen de novo . The inversion was verified with adaptive long-read sequencing (Oxford Nanopore Technologies, ONT), with 34 reads supporting the breakpoints. Breakpoint junction analysis revealed a one nucleotide microhomology and a 96 bp deletion (GRCh37, chr15:g.337,288,170 − 37,288,265) in BP1. In BP2 (GRCh37, chr15:g.41,663,130), located within a MER104, there was an 18 bp insertion, of which 11 bp were templated from the adjacent sequence of the inverted segment (Fig. 1 ). Transcriptome analysis revealed reduced expression of NUSAP1 (fold change 0.61) and no significant change in overall MEIS2 expression. Additionally, three fusion transcripts were found. One between the 3' end of MEIS2 exon 8 and the 5' end of NUSAP1 exon 8, and the others between the 3' end of NUSAP1 exon 8 and the 5' ends MEIS2 exons 9 and 10 respectively. These were confirmed by Sanger sequencing of cDNA (Fig. 2 ). DISCUSSION We report a balanced inversion of chromosome 15, inv(15)(q14q15), detected by short-read genome sequencing and confirmed using adaptive long-read sequencing, in a female proband with cleft palate and high functioning autism. The inversion disrupted two genes, MEIS2 and NUSAP1 , and resulted in at least three fusion transcripts. Long reads spanning the breakpoints allowed a detailed characterization of the rearrangement, which was not possible with short-read genome sequencing due to repetitive sequences (Fig. 1 ). The inversion breakpoints show evidence of a replicative error, specifically fork stalling and template switching (FoSTeS), characterized by base deletions and templated insertions (28). MEIS2 is linked to cleft palate, cardiac defects, and impaired intellectual development (CPCMR, MIM# 600987), whereas NUSAP1 has been described as tolerant of loss-of-function. However, a recent publication reported an NUSAP1 nonsense variant in two individuals with microcephaly, severe developmental delay, brain abnormalities, and seizures (29). A truncated transcript was detected that was hypothesized to evade nonsense mediated decay and have a toxic effect. In the proband presented here there is a clear clinical overlap with that of CPCMR. The presence of a likely non-functional MEIS2-NUSAP1 fusion transcript, along with the normal functional transcript, could be misinterpreted as two normal transcripts, potentially masking the effects of a functional haploinsufficiency. The milder phenotype, compared to previously reported MEIS2 cases, may indicate some remaining function in the product of the fusion gene. However, we cannot rule out a contribution from NUSAP1 . The identified NUSAP1-MEIS2 fusion transcripts could have a similar toxic effect as the truncated NUSAP1 transcript described above (29). In conclusion, by applying short-read genome sequencing, adaptive long-read and transcriptome analysis a cryptic Chromosome 15 inversion was investigated. The disease-causing rearrangement, undetected by all standard clinical diagnostic tests, showcase how balanced structural variants need to be included in genetic diagnostics of syndromes and neurodevelopmental disorders. Further studies are needed to determine the clinical impact of fusion transcripts in rare diseases. MATERIALS AND METHODS Ethics declaration The study was conducted in accordance with the Declaration of Helsinki and approved by the Regional Ethical Review Board in Stockholm, Sweden (protocol number 2019–04746). Written informed consent was provided by the participant and her parents to publish this paper. DNA and RNA extraction Genomic DNA was extracted from whole blood using the QIAsymphony instrument (QIAGEN, Hilden, Germany) with the QIAsymphony DSP DNA Midi Kit (cat. no. 937255, QIAGEN, Hilden, Germany), following the manufacturer's standard protocol. Fibroblasts were cultured from a skin biopsy in a medium composed of RPMI 1640 (1x) and Ham’s F-10 Nutrient Mixture at a 1:1 ratio, supplemented with 10% fetal bovine serum, 1% L-glutamine, and 0.2% Penicillin-Streptomycin solution (5000 U/mL). The cells were harvested by mechanical scraping from the culture flask and transferred to a PAXgene tube, and RNA was extracted using the PAXgene Blood RNA Kit (Preanalytix, cat. no. 762174, QIAGEN, Hilden, Germany) on a QIAcube Connect MDx system, following the standard protocol. The RNA was reverse transcribed to cDNA using SuperScript VILO Master Mix (QIAGEN, Hilden, Germany). The RNA template and nuclease-free water were added to the master mix and incubated in a thermal cycler at 25°C for 10 minutes, 42°C for 60 minutes, and 85°C for 5 minutes, followed by a hold at 4°C. Nuclease-free water was then added, and the cDNA was stored at -20°C. Genetic analysis Our clinical short-read genome sequencing workflow has been previously described (30–32). To verify the inversion call, we performed targeted long-read sequencing using the PromethION platform (ONT). The target region, spanning 32.5 Mb ([GRCh38] chr15:22,874,354 − 55,370,932) encompassed the entire inversion along with a 14 Mb buffer zone upstream and downstream of the region. Libraries were prepared from 2.76 µg of genomic DNA, following the ONT protocol 'Ligation Sequencing gDNA (SQK-LSK114), with an average sample fragment size of 50,031 bp. The fragment size was estimated using Femto Pulse, following the protocol for the 'Genomic DNA 165 kb Kit.' A single PromethION R10.4 (FLO-PRO114M) flow cell was used for sequencing. The base calling was performed using the Dorado basecaller ( https://github.com/nanoporetech/dorado ), which was run in High Accuracy Mode (HAC). The resulting data was processed using PoorPipe ( https://github.com/J35P312/poorpipe ), which performs alignment using Minimap2 (33), and SV calling using Sniffles 1 (34).The inversion was manually inspected in the Integrative Genomics Viewer (IGV) using the hg19/GRCh37 reference genome. Repetitive elements were analyzed using RepeatMasker in the UCSC Genome Browser. The effects on RNA were evaluated by whole-transcriptome sequencing of RNA isolated from cultured fibroblasts. Briefly, RNA was quantified and processed using a stranded, poly(A)-tailed kit (Illumina) before being subjected to 150 bp paired-end sequencing with approximately 150 million reads generated per sample on the Nova Seq X platform. The data was processed using the genomic medicine Sweden transcriptome pipeline Tomte ( https://github.com/genomic-medicine-sweden/tomte ). Briefly, the data was aligned to GRCh37 using STAR (35), next aberrant expression events were detected by Detection of RNA Outlier Pipeline (DROP) (36) using the default, recommended settings for OUTRIDER (37), and fusion transcripts were detected using STAR-Fusion (38). PCR and Sanger sequencing of fusion transcripts Primers targeting the inversion breakpoints in MEIS2 (exons 7 and 11) and NUSAP1 (exons 7 and 8) were designed (Supplementary Table S1 ). Breakpoint PCR was performed using AmpliTaq Gold (Fisher Scientific, Waltham, MA, USA) with a master mix containing PCR buffer II (1x), MgCl2 (2 mM), dNTPs (100 µM), and AmpliTaq Gold (1 U). The PCR conditions included 10 minutes at 96°C, followed by 35 cycles of 96°C (30 sec), 62°C (30 sec), and 72°C (2 min), with a final extension at 72°C for 10 minutes. Amplified products were detected using the FlashGel system (Lonza) with a 100 bp–3 kb DNA marker (cat. no. 57034, Lonza) and imaged on the GenoPlex system (VWR). Sanger sequencing was performed on the normal alleles of MEIS2 and NUSAP1 , and the fusion transcripts MEIS2-NUSAP1 and NUSAP1-MEIS2 (Supplementary Table S1 ). The PCR products were purified using Illustra ExoProStar, and the sequencing reaction was conducted with BigDye Terminator v3.1 (Applied Biosystems). Sequencing was done on an ABI 3500xL Genetic Analyzer (Applied Biosystems, Waltham, MA, USA). Declarations Competing interest Britt-Marie Anderlid, Jesper Eisfeldt, Marlene Ek, Maria Johansson Soller, Malin Kvarnung, Anna Lindstrand, Maria Pettersson, and Håkan Thonberg declare no potential conflict of interest. Funding This work was supported by the Swedish Research Council [2019–02078], the Swedish Brain Fund [FO2022-0256], the Stockholm City Council and the Swedish Rare Diseases Research Foundation (Sällsyntafonden). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contribution A.L. conceptualized the study. B-M.A. wrote the phenotype description and performed the medical investigations. M.P., J.E. and M.E. did the genomic analysis and J.E., H.T. and M.E did the analysis of the transcriptome. A.L., M.K. and M.J.S evaluated the findings. M.E. designed the figures. M.E. wrote the original draft, and all the other authors contributed to the final manuscript. A.L. acquired funding for the project. Acknowledgement The authors acknowledge support from the National Genomics Infrastructure in Stockholm funded by Science for Life Laboratory, the Knut and Alice Wallenberg Foundation and the Swedish Research Council, and the NAISS/Uppsala Multidisciplinary Center for Advanced Computational Science for assistance with massively parallel sequencing and access to the UPPMAX computational infrastructure. 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Additional Declarations No competing interests reported. Supplementary Files SupplementaryTableS1.docx Cite Share Download PDF Status: Published Journal Publication published 05 Dec, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 28 Oct, 2024 Reviews received at journal 25 Oct, 2024 Reviews received at journal 14 Oct, 2024 Reviewers agreed at journal 13 Oct, 2024 Reviewers agreed at journal 11 Oct, 2024 Reviewers invited by journal 11 Oct, 2024 Editor assigned by journal 11 Oct, 2024 Editor invited by journal 11 Oct, 2024 Submission checks completed at journal 10 Oct, 2024 First submitted to journal 18 Sep, 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. 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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-5112053","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":365453510,"identity":"acc101ed-24a3-484a-be09-5acbda975b5f","order_by":0,"name":"Marlene Ek","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIie3SMQrCMBiG4S8IThHXSKE9gRARpEPRqwiFungGKQjportOXsMxJaBLxLVjJ+e61UE0iK5GcXHIuyTD//BnCOBy/WdEPg4KUlaSfWZepNFbfUuaHpUfjHezQynP2whtM+5FOkSQpe/JQE94vtYJOssU/WnBwLVl1UAmUC2hwI9APK0MYWMLOZ6gruKGkSEqNCTYlBZSmC1ESPBWSuYwD0PxXhhyQr4UMWU6n5OFZpRr68MSUl7E0G8v4n1d72Z+kEnLmmfU/IL0eXG5XC7Xz90BYpNEZiKTKwUAAAAASUVORK5CYII=","orcid":"","institution":"Department of Molecular Medicine and Surgery, Karolinska Institutet","correspondingAuthor":true,"prefix":"","firstName":"Marlene","middleName":"","lastName":"Ek","suffix":""},{"id":365453511,"identity":"05b22f9a-fdd0-4ecc-8f6c-c87ddb0fde00","order_by":1,"name":"Malin Kvarnung","email":"","orcid":"","institution":"Department of Molecular Medicine and Surgery, Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Malin","middleName":"","lastName":"Kvarnung","suffix":""},{"id":365453512,"identity":"bb7f4200-f575-4438-a3a1-fed524fd8625","order_by":2,"name":"Maria Pettersson","email":"","orcid":"","institution":"Department of Molecular Medicine and Surgery, Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Pettersson","suffix":""},{"id":365453513,"identity":"60b26d02-0e9b-42e2-bf46-ba203bf30533","order_by":3,"name":"Maria Johansson Soller","email":"","orcid":"","institution":"Department of Molecular Medicine and Surgery, Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Johansson","lastName":"Soller","suffix":""},{"id":365453514,"identity":"98970fc5-f9e2-4a04-b9f2-9eded504e6da","order_by":4,"name":"Britt-Marie Anderlid","email":"","orcid":"","institution":"Department of Molecular Medicine and Surgery, Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Britt-Marie","middleName":"","lastName":"Anderlid","suffix":""},{"id":365453515,"identity":"fd429c9f-8205-47aa-ab28-9132dc77fb32","order_by":5,"name":"Håkan Thonberg","email":"","orcid":"","institution":"Department of Molecular Medicine and Surgery, Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Håkan","middleName":"","lastName":"Thonberg","suffix":""},{"id":365453516,"identity":"69da4ddc-e2c8-4d2d-ad28-aa2729107ff8","order_by":6,"name":"Jesper Eisfeldt","email":"","orcid":"","institution":"Department of Clinical Genetics and Genomics, Karolinska University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jesper","middleName":"","lastName":"Eisfeldt","suffix":""},{"id":365453517,"identity":"fc594814-d13a-41a6-bd23-570154aca13f","order_by":7,"name":"Anna Lindstrand","email":"","orcid":"","institution":"Department of Molecular Medicine and Surgery, Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Lindstrand","suffix":""}],"badges":[],"createdAt":"2024-09-18 18:42:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5112053/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5112053/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-81507-7","type":"published","date":"2024-12-05T15:58:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":66557550,"identity":"caa8b8ec-dc32-4ffc-a768-c6db5004b804","added_by":"auto","created_at":"2024-10-14 09:51:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1213539,"visible":true,"origin":"","legend":"\u003cp\u003eVisualization of paracentric inversion of Chromosome 15 including breakpoint analysis. Breakpoint 1 (BP1) at cytoband 15q14 disrupts MEIS2 and breakpoint 2 (BP2) at cytoband 15q15 disrupts \u003cem\u003eNUSAP1\u003c/em\u003e. Repetitive elements are shown in blue boxes. Short-read genome sequencing spanning the breakpoints displays mate-pairs in teal and dark blue, respectively. Reads with rainbow pattern from long-read genome sequencing data indicate the two breakpoints. The breakpoint analysis with genomic positioning in GRCh19. Deleted nucleotides are shown in lower case red letters, microhomology in bold purple letters, and the inserted nucleotides in bold magenta letters.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5112053/v1/8dd40c860043f704bdf97349.png"},{"id":66559121,"identity":"02ecc774-f3c1-46cc-9972-87366cd5c00a","added_by":"auto","created_at":"2024-10-14 09:59:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1655199,"visible":true,"origin":"","legend":"\u003cp\u003eDepiction of fusion transcripts identified in our proband. \u003cem\u003eMEIS2\u003c/em\u003e transcript (NM_170675.5) depicted in blue, \u003cem\u003eNUSAP1\u003c/em\u003e transcript (NM_016359.5) in red. (a) Fusion transcript of \u003cem\u003eMEIS2\u003c/em\u003e-\u003cem\u003eNUSAP1\u003c/em\u003ewith Sanger sequencing across the fusion junction (dashed line) of exon 8 of \u003cem\u003eMEIS2\u003c/em\u003eand exon 8 of \u003cem\u003eNUSAP1\u003c/em\u003e. (b) Fusion transcripts of \u003cem\u003eNUSAP1\u003c/em\u003e-\u003cem\u003eMEIS2\u003c/em\u003ewith Sanger sequencing across the fusion junction (dashed line) of exon 7 of \u003cem\u003eNUSAP1\u003c/em\u003eand exon 9 of \u003cem\u003eMEIS2\u003c/em\u003e, as well as exon 7 of \u003cem\u003eNUSAP1\u003c/em\u003e and exon 10 of \u003cem\u003eMEIS2\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5112053/v1/78e4d34ada694551579ae3e5.png"},{"id":70965508,"identity":"4c586d2f-f675-4550-94bb-292b8aeb2f6e","added_by":"auto","created_at":"2024-12-09 16:20:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2801732,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5112053/v1/06b0f1fc-1056-4e5d-86fe-4f90ccbc4abe.pdf"},{"id":66557552,"identity":"52ecb644-4db3-4b79-b561-3c35e44516c7","added_by":"auto","created_at":"2024-10-14 09:51:46","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15222,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5112053/v1/42d774405ddbea814c5a2b47.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Adaptive long-read and transcriptome sequencing detail a submicroscopic inv(15)(q14q15), generating fusion transcripts and MEIS2 and NUSAP1 haploinsufficiency","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePathogenic variants in \u003cem\u003eMEIS2\u003c/em\u003e are associated with a syndrome known as \u0026ldquo;cleft palate, cardiac defects, and impaired intellectual development\u0026rdquo; (CPCMR, MIM# 600987). \u003cem\u003eMEIS2\u003c/em\u003e, a homeodomain-containing protein, is part of the three amino acid loop extension (TALE) family, and functions as a transcription regulator. \u003cem\u003eMeis2\u003c/em\u003e has been implicated in the development of limbs and brain in chicks, lens and retina formation in mice and \u003cem\u003eMekada\u003c/em\u003e fish, and is essential for both cardiac and neural crest development in mice. (1\u0026ndash;5) The gene is highly intolerant to loss-of-function mutations, with a pLI score greater than 0.9, and causes disease primarily through haploinsufficiency. Most of the described \u003cem\u003eMEIS2\u003c/em\u003e variants are \u003cem\u003ede novo\u003c/em\u003e, although cases of inherited variants have been described. Carrier parents tend to exhibit a similar phenotype, with mosaic carriers showing milder expression. (6\u0026ndash;10)\u003c/p\u003e \u003cp\u003eThe previously reported disease-causing variants in \u003cem\u003eMEIS2\u003c/em\u003e include both structural variants and sequence variants, such as single nucleotide variants (SNVs) and insertions/deletions (INDELs). The structural variants are predominantly copy number variants (CNVs), with deletions, ranging in size from 0.6 to 6.97 Mb, and one family reported with a 58 kb tandem duplication. One of the deletions was part of a \u003cem\u003ede novo\u003c/em\u003e complex chromosomal rearrangement. (6\u0026ndash;8, 11\u0026ndash;18) Notably, one case involving a balanced structural variant has been reported: a balanced reciprocal translocation between chromosomes 11 and 15, t(11;15)(p14;q14), where the breakpoint on chromosome 15 disrupts \u003cem\u003eMEIS2\u003c/em\u003e in intron 6. (19) The reported SNV/INDELs include truncating, splice site, missense variants or in-frame deletions within the homeodomain. (9, 10, 18, 20\u0026ndash;27)\u003c/p\u003e \u003cp\u003eWe detail the genetic aberrations in a proband with cleft palate and high-functioning autism. Structural variant analysis of short-read genome sequencing revealed a 4.37 Mb inversion on chromosome 15, with breakpoints in intron 8 of \u003cem\u003eMEIS2\u003c/em\u003e and intron 7 of \u003cem\u003eNUSAP1\u003c/em\u003e, generating two fusion transcripts.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eThe proband is the first child of healthy, non-consanguineous parents. She was born after an uneventful pregnancy at 39 weeks\u0026thinsp;+\u0026thinsp;6 days (birth weight 3670 g, birth length 53 cm and head circumference 35 cm). A posterior cleft palate was diagnosed early, and she experienced feeding difficulties during the first months of life. The cleft palate was surgically corrected twice, at 5 and 12 months of age. Her motor development was delayed; she sat unsupported at 9 months and walked independently at 19 months of age. She exhibited hypotonia and hypermobile joints, and her postnatal growth was slightly restricted, with a head circumference of -2 SD and a length of -0,5 SD at 3 years of age. Adhesions between labia majora initially raised suspicion of urethral duplication, but an ultrasound of the urinary tracts was normal. A cardiac ultrasound revealed a small atrial septal defect, which was clinically insignificant. A neuropsychological evaluation at the age of 5.5 years diagnosed her with high-function autism and her intellectual level was within the normal range. Some facial features were present, including penciled eyebrows, rounded eyes, large and protruding ears, and discrete fetal finger pads. She has two younger, healthy siblings. Genetic tests included array comparative genomic hybridization at 18 months of age, short-read genome sequencing analysis with \u003cem\u003ein-silico\u003c/em\u003e gene panels for neuromuscular disorders (499 genes) and intellectual disability (885 genes) at age 7, all with normal results.\u003c/p\u003e \u003cp\u003eGenome-wide structural variant analysis of the short-read genome sequencing data identified a 4.37 Mb inversion on chromosome 15, with breakpoint 1 (BP1), localized in intron 8 of the MANE transcript NM_170675.5 of \u003cem\u003eMEIS2\u003c/em\u003e and breakpoint 2 (BP2) in intron 7 of \u003cem\u003eNUSAP1\u003c/em\u003e (NM_016359.5). Follow-up analysis of the parents confirmed that the variant had arisen \u003cem\u003ede novo\u003c/em\u003e. The inversion was verified with adaptive long-read sequencing (Oxford Nanopore Technologies, ONT), with 34 reads supporting the breakpoints. Breakpoint junction analysis revealed a one nucleotide microhomology and a 96 bp deletion (GRCh37, chr15:g.337,288,170\u0026thinsp;\u0026minus;\u0026thinsp;37,288,265) in BP1. In BP2 (GRCh37, chr15:g.41,663,130), located within a MER104, there was an 18 bp insertion, of which 11 bp were templated from the adjacent sequence of the inverted segment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTranscriptome analysis revealed reduced expression of \u003cem\u003eNUSAP1\u003c/em\u003e (fold change 0.61) and no significant change in overall \u003cem\u003eMEIS2\u003c/em\u003e expression. Additionally, three fusion transcripts were found. One between the 3' end of \u003cem\u003eMEIS2\u003c/em\u003e exon 8 and the 5' end of \u003cem\u003eNUSAP1\u003c/em\u003e exon 8, and the others between the 3' end of \u003cem\u003eNUSAP1\u003c/em\u003e exon 8 and the 5' ends \u003cem\u003eMEIS2\u003c/em\u003e exons 9 and 10 respectively. These were confirmed by Sanger sequencing of cDNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eWe report a balanced inversion of chromosome 15, inv(15)(q14q15), detected by short-read genome sequencing and confirmed using adaptive long-read sequencing, in a female proband with cleft palate and high functioning autism. The inversion disrupted two genes, \u003cem\u003eMEIS2\u003c/em\u003e and \u003cem\u003eNUSAP1\u003c/em\u003e, and resulted in at least three fusion transcripts.\u003c/p\u003e \u003cp\u003eLong reads spanning the breakpoints allowed a detailed characterization of the rearrangement, which was not possible with short-read genome sequencing due to repetitive sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The inversion breakpoints show evidence of a replicative error, specifically fork stalling and template switching (FoSTeS), characterized by base deletions and templated insertions (28).\u003c/p\u003e \u003cp\u003e \u003cem\u003eMEIS2\u003c/em\u003e is linked to cleft palate, cardiac defects, and impaired intellectual development (CPCMR, MIM# 600987), whereas \u003cem\u003eNUSAP1\u003c/em\u003e has been described as tolerant of loss-of-function. However, a recent publication reported an \u003cem\u003eNUSAP1\u003c/em\u003e nonsense variant in two individuals with microcephaly, severe developmental delay, brain abnormalities, and seizures (29). A truncated transcript was detected that was hypothesized to evade nonsense mediated decay and have a toxic effect. In the proband presented here there is a clear clinical overlap with that of CPCMR. The presence of a likely non-functional \u003cem\u003eMEIS2-NUSAP1\u003c/em\u003e fusion transcript, along with the normal functional transcript, could be misinterpreted as two normal transcripts, potentially masking the effects of a functional haploinsufficiency. The milder phenotype, compared to previously reported \u003cem\u003eMEIS2\u003c/em\u003e cases, may indicate some remaining function in the product of the fusion gene. However, we cannot rule out a contribution from \u003cem\u003eNUSAP1\u003c/em\u003e. The identified \u003cem\u003eNUSAP1-MEIS2\u003c/em\u003e fusion transcripts could have a similar toxic effect as the truncated \u003cem\u003eNUSAP1\u003c/em\u003e transcript described above (29).\u003c/p\u003e \u003cp\u003eIn conclusion, by applying short-read genome sequencing, adaptive long-read and transcriptome analysis a cryptic Chromosome 15 inversion was investigated. The disease-causing rearrangement, undetected by all standard clinical diagnostic tests, showcase how balanced structural variants need to be included in genetic diagnostics of syndromes and neurodevelopmental disorders. Further studies are needed to determine the clinical impact of fusion transcripts in rare diseases.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eEthics declaration\u003c/h2\u003e \u003cp\u003eThe study was conducted in accordance with the Declaration of Helsinki and approved by the Regional Ethical Review Board in Stockholm, Sweden (protocol number 2019\u0026ndash;04746).\u003c/p\u003e \u003cp\u003eWritten informed consent was provided by the participant and her parents to publish this paper.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDNA and RNA extraction\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was extracted from whole blood using the QIAsymphony instrument (QIAGEN, Hilden, Germany) with the QIAsymphony DSP DNA Midi Kit (cat. no. 937255, QIAGEN, Hilden, Germany), following the manufacturer's standard protocol.\u003c/p\u003e \u003cp\u003eFibroblasts were cultured from a skin biopsy in a medium composed of RPMI 1640 (1x) and Ham\u0026rsquo;s F-10 Nutrient Mixture at a 1:1 ratio, supplemented with 10% fetal bovine serum, 1% L-glutamine, and 0.2% Penicillin-Streptomycin solution (5000 U/mL). The cells were harvested by mechanical scraping from the culture flask and transferred to a PAXgene tube, and RNA was extracted using the PAXgene Blood RNA Kit (Preanalytix, cat. no. 762174, QIAGEN, Hilden, Germany) on a QIAcube Connect MDx system, following the standard protocol.\u003c/p\u003e \u003cp\u003eThe RNA was reverse transcribed to cDNA using SuperScript VILO Master Mix (QIAGEN, Hilden, Germany). The RNA template and nuclease-free water were added to the master mix and incubated in a thermal cycler at 25\u0026deg;C for 10 minutes, 42\u0026deg;C for 60 minutes, and 85\u0026deg;C for 5 minutes, followed by a hold at 4\u0026deg;C. Nuclease-free water was then added, and the cDNA was stored at -20\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eGenetic analysis\u003c/h3\u003e\n\u003cp\u003eOur clinical short-read genome sequencing workflow has been previously described (30\u0026ndash;32). To verify the inversion call, we performed targeted long-read sequencing using the PromethION platform (ONT). The target region, spanning 32.5 Mb ([GRCh38] chr15:22,874,354\u0026thinsp;\u0026minus;\u0026thinsp;55,370,932) encompassed the entire inversion along with a 14 Mb buffer zone upstream and downstream of the region. Libraries were prepared from 2.76 \u0026micro;g of genomic DNA, following the ONT protocol 'Ligation Sequencing gDNA (SQK-LSK114), with an average sample fragment size of 50,031 bp. The fragment size was estimated using Femto Pulse, following the protocol for the 'Genomic DNA 165 kb Kit.' A single PromethION R10.4 (FLO-PRO114M) flow cell was used for sequencing. The base calling was performed using the Dorado basecaller (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/nanoporetech/dorado\u003c/span\u003e\u003cspan address=\"https://github.com/nanoporetech/dorado\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which was run in High Accuracy Mode (HAC). The resulting data was processed using PoorPipe (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/J35P312/poorpipe\u003c/span\u003e\u003cspan address=\"https://github.com/J35P312/poorpipe\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which performs alignment using Minimap2 (33), and SV calling using Sniffles 1 (34).The inversion was manually inspected in the Integrative Genomics Viewer (IGV) using the hg19/GRCh37 reference genome. Repetitive elements were analyzed using RepeatMasker in the UCSC Genome Browser.\u003c/p\u003e \u003cp\u003eThe effects on RNA were evaluated by whole-transcriptome sequencing of RNA isolated from cultured fibroblasts. Briefly, RNA was quantified and processed using a stranded, poly(A)-tailed kit (Illumina) before being subjected to 150 bp paired-end sequencing with approximately 150\u0026nbsp;million reads generated per sample on the Nova Seq X platform. The data was processed using the genomic medicine Sweden transcriptome pipeline Tomte (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/genomic-medicine-sweden/tomte\u003c/span\u003e\u003cspan address=\"https://github.com/genomic-medicine-sweden/tomte\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Briefly, the data was aligned to GRCh37 using STAR (35), next aberrant expression events were detected by Detection of RNA Outlier Pipeline (DROP) (36) using the default, recommended settings for OUTRIDER (37), and fusion transcripts were detected using STAR-Fusion (38).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePCR and Sanger sequencing of fusion transcripts\u003c/h2\u003e \u003cp\u003ePrimers targeting the inversion breakpoints in \u003cem\u003eMEIS2\u003c/em\u003e (exons 7 and 11) and \u003cem\u003eNUSAP1\u003c/em\u003e (exons 7 and 8) were designed (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Breakpoint PCR was performed using AmpliTaq Gold (Fisher Scientific, Waltham, MA, USA) with a master mix containing PCR buffer II (1x), MgCl2 (2 mM), dNTPs (100 \u0026micro;M), and AmpliTaq Gold (1 U). The PCR conditions included 10 minutes at 96\u0026deg;C, followed by 35 cycles of 96\u0026deg;C (30 sec), 62\u0026deg;C (30 sec), and 72\u0026deg;C (2 min), with a final extension at 72\u0026deg;C for 10 minutes.\u003c/p\u003e \u003cp\u003eAmplified products were detected using the FlashGel system (Lonza) with a 100 bp\u0026ndash;3 kb DNA marker (cat. no. 57034, Lonza) and imaged on the GenoPlex system (VWR). Sanger sequencing was performed on the normal alleles of \u003cem\u003eMEIS2\u003c/em\u003e and \u003cem\u003eNUSAP1\u003c/em\u003e, and the fusion transcripts \u003cem\u003eMEIS2-NUSAP1\u003c/em\u003e and \u003cem\u003eNUSAP1-MEIS2\u003c/em\u003e (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe PCR products were purified using Illustra ExoProStar, and the sequencing reaction was conducted with BigDye Terminator v3.1 (Applied Biosystems). Sequencing was done on an ABI 3500xL Genetic Analyzer (Applied Biosystems, Waltham, MA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interest\u003c/h2\u003e \u003cp\u003eBritt-Marie Anderlid, Jesper Eisfeldt, Marlene Ek, Maria Johansson Soller, Malin Kvarnung, Anna Lindstrand, Maria Pettersson, and H\u0026aring;kan Thonberg declare no potential conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Swedish Research Council [2019\u0026ndash;02078], the Swedish Brain Fund [FO2022-0256], the Stockholm City Council and the Swedish Rare Diseases Research Foundation (S\u0026auml;llsyntafonden). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.L. conceptualized the study. B-M.A. wrote the phenotype description and performed the medical investigations. M.P., J.E. and M.E. did the genomic analysis and J.E., H.T. and M.E did the analysis of the transcriptome. A.L., M.K. and M.J.S evaluated the findings. M.E. designed the figures. M.E. wrote the original draft, and all the other authors contributed to the final manuscript. A.L. acquired funding for the project.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors acknowledge support from the National Genomics Infrastructure in Stockholm funded by Science for Life Laboratory, the Knut and Alice Wallenberg Foundation and the Swedish Research Council, and the NAISS/Uppsala Multidisciplinary Center for Advanced Computational Science for assistance with massively parallel sequencing and access to the UPPMAX computational infrastructure.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe BAM files from the adaptive long-read sequencing is submitted to the European Genome-phenome Archive (EGA) web portal (https://ega-archive.org/datasets/EGAD50000000676) under submission ID 1507.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eCapdevila J, Tsukui T, Rodr\u0026iacute;quez Esteban C, Zappavigna V, Izpis\u0026uacute;a Belmonte JC. 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Accuracy assessment of fusion transcript detection via read-mapping and de novo fusion transcript assembly-based methods. Genome Biol. 2019;20(1):213.\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Inversion, adaptive long-read genome sequencing, RNA sequencing, MEIS2, NUSAP1, gene disruption","lastPublishedDoi":"10.21203/rs.3.rs-5112053/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5112053/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInversions are balanced structural variants that often remain undetected in genetic diagnostics. We present a female proband with a \u003cem\u003ede novo\u003c/em\u003e Chromosome 15 paracentric inversion, disrupting \u003cem\u003eMEIS2\u003c/em\u003e and \u003cem\u003eNUSAP1\u003c/em\u003e. The inversion was detected by short-read genome sequencing and confirmed with adaptive long-read sequencing. The breakpoint junction analysis revealed a 96 base pair (bp) deletion and an 18 bp insertion in the two junctions, suggesting that the rearrangement arose through a replicative error. Transcriptome sequencing of cultured fibroblasts revealed normal \u003cem\u003eMEIS2\u003c/em\u003e levels and 0.61-fold decreased expression of\u003cem\u003e NUSAP1\u003c/em\u003e. Furthermore, two fusion transcripts were detected and confirmed by Sanger sequencing.\u003c/p\u003e\n\u003cp\u003eHeterozygous loss of \u003cem\u003eMEIS2\u003c/em\u003e (MIM# 600987) is associated with a cleft palate, heart malformations, and intellectual impairment, which overlap with the clinical symptoms observed in the proband. The observed fusion transcripts are likely non-functional, and \u003cem\u003eMEIS2\u003c/em\u003ehaploinsufficiency is the likely disease causative mechanism. Altogether, this study findings illustrate the importance of including inversions in rare disease diagnostic testing and highlight the value of long read sequencing for the validation and characterization of such variants.\u003c/p\u003e","manuscriptTitle":"Adaptive long-read and transcriptome sequencing detail a submicroscopic inv(15)(q14q15), generating fusion transcripts and MEIS2 and NUSAP1 haploinsufficiency","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-14 09:51:41","doi":"10.21203/rs.3.rs-5112053/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-28T09:55:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-25T15:19:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-14T19:53:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"133813939047413033030057746623018481537","date":"2024-10-13T16:22:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"258791867603881883766952660040246100617","date":"2024-10-11T16:53:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-11T15:56:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-11T15:55:07+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-10-11T11:57:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-10T10:09:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-09-18T18:24:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"df04d3d6-56c9-4ba2-be11-2fdaba1d368f","owner":[],"postedDate":"October 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":38878530,"name":"Health sciences/Medical research"},{"id":38878531,"name":"Health sciences/Molecular medicine"},{"id":38878532,"name":"Health sciences/Neurology"},{"id":38878533,"name":"Health sciences/Diseases/Neurological disorders"}],"tags":[],"updatedAt":"2024-12-09T16:17:07+00:00","versionOfRecord":{"articleIdentity":"rs-5112053","link":"https://doi.org/10.1038/s41598-024-81507-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-12-05 15:58:14","publishedOnDateReadable":"December 5th, 2024"},"versionCreatedAt":"2024-10-14 09:51:41","video":"","vorDoi":"10.1038/s41598-024-81507-7","vorDoiUrl":"https://doi.org/10.1038/s41598-024-81507-7","workflowStages":[]},"version":"v1","identity":"rs-5112053","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5112053","identity":"rs-5112053","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}