Long-read sequencing reveals a hidden Alu-mediated splice defect in CPLANE1, causing orofaciodigital syndrome type VI

Long-read sequencing reveals a hidden Alu-mediated splice defect in CPLANE1, causing orofaciodigital syndrome type VI | 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 Brief Communication Long-read sequencing reveals a hidden Alu-mediated splice defect in CPLANE1, causing orofaciodigital syndrome type VI Malte Spielmann, Jelena Pozojevic, Henrike Sczakiel, Saranya Balachandran, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9094779/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Orofaciodigital syndrome type VI (OFD VI) is a recessive ciliopathy characterized by excessive polydactyly, molar tooth sign, cleft lip, and developmental delay, caused by pathogenic variants in CPLANE1 . Here, we present a patient with OFD VI that remained genetically unexplained after routine genetic testing, including short-read whole genome sequencing (WGS). Using long-read sequencing, we found two biallelic splice-site variants in CPLANE1 , c.8633-4_8633-3del, and an Alu element insertion close to an exon-intron boundary. Transcript analysis showed that each variant independently resulted in exon skipping, and quantitative expression studies revealed reduced total CPLANE1 mRNA levels in patient-derived fibroblasts. Based on these findings, we were able to re-classify the c.8633-4_8633-3del variant from a variant of uncertain significance (VUS) to pathogenic. The identification of an Alu element insertion missed by short-read WGS highlights the added diagnostic value of long-read sequencing in uncovering cryptic, transposable element-associated pathogenic variants. Health sciences/Medical research/Genetics research Biological sciences/Genetics/Gene expression Figures Figure 1 Figure 2 Figure 3 Introduction Next-generation sequencing (NGS) has transformed molecular diagnostics, and whole exome sequencing (WES) is widely used to detect protein-coding, disease-causing variants. However, whole genome sequencing (WGS) has the potential to replace multiple diagnostic methods such as chromosome analysis, array-CGH and WES by providing a single diagnostic test 1 . Despite their high accuracy, short-read sequencing technologies struggle to resolve complex genomic regions, including repetitive and GC-rich sequences. Long-read sequencing overcomes these limitations, enabling improved detections of structural variants, repeat expansions, and epigenetic modifications, as we recently showed on examples of the GGC-repeat expansion in ZFHX3 and changed DNA methylation due to a retrotransposon insertion 2 , 3 . Orofaciodigital syndrome type VI (OFD VI) (OMIM #277170) is an autosomal recessive ciliopathy that belongs to the Joubert syndrome related disorders. Diagnostic criteria for OFD VI include the molar tooth sign and one or more of the following: 1) tongue hamartoma(s) and/or additional frenula and/or upper lip notch; 2) mesoaxial polydactyly of one or more hands or feet; 3) hypothalamic hamartoma 4 . The primary gene responsible for OFD VI is CPLANE1 , (also called C5orf42), which encodes a protein involved in ciliogenesis, cell migration, and Hedgehog signaling 5 . Pathogenic variants in this gene are also linked to Joubert syndrome 17 (OMIM #614615) 6 , 7 . In this study, we present a patient initially diagnosed with excessive polydactyly, cleft lip, and abnormal tongue, with a family history of polydactyly. While short-read WGS failed to identify the genetic cause of the disease, long-read WGS revealed two biallelic splice-site variants in CPLANE1 . Further functional assays on patient-derived fibroblasts revealed pathogenic aberrant splicing effects and decreased CPLANE1 . Our work emphasizes the utility of long-read sequencing especially in detecting clinically relevant transposable element insertions, and re-classifies a rare splice-site variant present in the general population. Materials and Methods Cell culture and DNA extraction Following informed consent, patient-derived fibroblasts were obtained from a skin biopsy and cultured under standard conditions. High molecular weight DNA was extracted using MagAttract HMW DNA kit (Qiagen), according to the manufacturer's protocol. Long-read sequencing and data analysis PacBio HiFi long-read sequencing was performed on sheared genomic DNA. Libraries were size-selected to exclude fragments shorter than 10 kb and sequenced on a Sequel II platform. Reads were aligned to the GRCh38 reference genome, followed by variant calling and phasing. Variant interpretation was conducted using the Geneyx software, and visualization was performed with the integrated genomic viewer. RNA extraction and quantitative PCR (qPCR) RNA was extracted from patient-derived and control fibroblasts and reverse-transcribed into cDNA. Quantitative PCR (qPCR) was used to assess CPLANE1 expression levels relative to controls. Due to the single-patient design, results were evaluated descriptively without statistical testing. Sanger sequencing Targeted PCR amplification and Sanger sequencing were performed to validate identified variants and assess their effects on mRNA splicing. Results Clinical report At 1.5 years of age the male patient presented with postaxial polydactyly of both hands, polydactyly of the left foot, and polysyndactyly of the right foot (Fig. 1 A). The proband is the first son of healthy, non-consanguineous parents. Family history was positive for polydactyly, with father's cousin (child of consanguineous parents) also reported to show polydactyly. Additionally, the patient presented with a cleft lip and an abnormally shaped tongue. A follow-up evaluation at the age of 6 years additionally revealed global developmental delay (walking independently at the age of 3.5 years, still non-verbal at the current age of 6 years), muscular hypotonia, myopia and hearing impairment. Molecular genetics findings Short-read WGS did not detect the disease-causing variant(s) and thus the patient was selected for long-read WGS (LR-WGS). Long-read PacBio HiFi sequencing revealed two CPLANE1 variants as the most plausible cause of the phenotype: c.8633-4_8633-3del (NM_001384732.1) and an Alu element insertion at the exon 14/ intron 13 boundary. Phasing the reads showed that they are located on two alleles, consistent with recessive disease inheritance mode (Fig. 1 B). The c.8633-4_8633-3del is present in gnomAD v4.1 at allele frequency 0.00002653 and classified as a variant of unknown significance (VUS) both in gnomAD and ClinVar. In contrast, the Alu element insertion is not present in gnomAD, and the sequence analysis revealed that it belongs to the AluYa5 family, a young and highly active group of Alu elements. This Alu insertion is flanked by the target site duplication (TSD) sequence, a hallmark of retrotransposon insertions. Based on the TSD sequence, the genomic location of this Alu element insertion is left-normalized and defined to be chr5:37,224,330 (GRCh38). Functional assessments of the variants Since both these variants are intronic, close to the exon-intron boundaries, we next tested their pathogenic splicing potential. Sanger sequencing of mRNA extracted from patient-derived fibroblasts revealed that each of these variants leads to exon skipping: c.8633-4_8633-3del leads to exon 45 skipping (Fig. 2 A), while the AluYa5 insertion leads to exon 14 skipping (Fig. 2 B). Both events result in frameshift (exon 45 is 81 bp long, while exon 14 is 31 bp long), predicted to trigger nonsense-mediated mRNA decay (NMD). Quantitative PCR confirmed a substantial reduction of CPLANE1 mRNA levels in the patient compared to controls (Fig. 2 C), supporting biallelic loss-of-function. Re-analysis of the short-read WGS data did not identify the Alu insertion in either structural variant or single nucleotide variant call sets. However, subsequent inspection of the corresponding BAM files revealed soft-clipped reads at the insertion site (Fig. 3 ). Discussion Our patient was initially diagnosed with polydactyly, with positive family history, but received no molecular diagnosis even after WGS using short-read technologies. Because of this, the patient was selected for long-read WGS, which indeed detected a previously missed second hit in a recessive gene, an Alu element insertion. Phasing the long reads enabled us to show biallelic nature of the two CPLANE1 variants, while further functional validation demonstrated their pathogenic potential, resulting in loss-of-function. Our work demonstrated that the c.8633-4_8633-3del in intron 44, ultra rare in the general population, leads to exon 45 skipping and frameshift, allowing us to re-classify it as pathogenic (instead of VUS), according to the American College of Medical Genetics and Genomics (ACMG) criteria 8 , 9 . Similarly, the Alu element insertion close to the exon-intron boundary, absent in population databases, leads to exon 14 skipping and frameshift, and we classify this variant as pathogenic based on the ACMG criteria (PVS1, PS3, PM2, PM3). Further functional evidence that demonstrates pathogenicity of both variants includes markedly decreased CPLANE1 transcript levels in patient-derived fibroblasts, as detected by qPCR, consistent with frameshift variants predicted to undergo NMD. Of note, the molecular diagnosis by LR-WGS was based solely on the information that the patient presents with polydactyly, and the variant interpretation was based on the human phenotype ontology terms (HP:0010442, HP:0010689). Revisiting the phenotype at the age of 6 years showed that the patient presents with neurodevelopmental delay in addition to the initially observed polydactyly, which led the clinician to suspect OFD syndrome. Thus, the molecular diagnosis is in accordance with the clinical diagnosis, establishing that the patient presents with OFD VI. The Alu insertion was not detected by standard variant calling approaches applied to short-read WGS data, highlighting a limitation of this methodology for identifying transposable element insertions. Its detection by PacBio long-read sequencing underscores the added value of long-read technologies in resolving such variants. Retrospective examination of the short-read alignments revealed soft-clipped reads at the locus, suggesting that while signals of the insertion were present, they were not captured by conventional variant calling pipelines. Transposable elements (TEs) constitute nearly 50% of the human genome. While many of them are evolutionary ancient and inactive, several families remain active and capable of mobilization, most notably Alu, LINE, and SVA elements. As a result, they contribute both to phenotypic diversity and human disease. Despite their biological and clinical relevance, TEs are frequently underdetected by conventional sequencing approaches, including short-read technologies. This limitation arises in part from their highly repetitive nature and widespread genomic distribution, which complicates accurate read mapping and insertion site resolution. Additionally, many TEs are GC-rich, leading to amplification bias and reduced coverage due to inefficient polymerase activity during amplification. Consequently, the full extent of their variation and activity remains incompletely characterized using standard sequencing methods. AluYa5 family is the youngest and most active among Alu elements, and has been associated with human disease, including neurodevelopmental disorders and cancer 10 , 11 , 12 . Insertions within introns or regulatory regions can disrupt gene expression through altered splicing, premature polyadenylation, or transcriptional interference, depending on the location of their insertion and the genomic context 13 , 14 . It is tempting to speculate whether the Alu element identified in our patient is a de novo insertion or is absent in population databases due to limitations of short-read sequencing technologies. In conclusion, as LR-WGS enters clinical diagnostics, our work highlights its clear technical advantages in mapping accuracy, sequence resolution, and variant phasing — without requiring parental samples or additional downstream analyses. Beyond this, LR-WGS offers added strengths, including integrated epigenetic profiling and the potential to function as a single, comprehensive diagnostic test. Moreover, LR-WGS promises to provide deeper insight into the non-coding genome, enabling the identification of previously undetected regulatory variants and disease mechanisms 15 . Declarations Acknowledgements The authors are grateful to the patient and his family for participating in this study. Author Contribution Statement JP analyzed LR-WGS data, performed the functional assays, and wrote the first draft of the manuscript. HLS performed clinical assessment of the patient and edited the manuscript. NK prepared the sample for LR-WGS. SB performed bioinformatic analyses. MAM performed clinical assessment of the patient. WH performed surgery and took the skin specimen. KH supervised the LR-WGS and edited the manuscript. MS supervised the entire project and edited the manuscript. Funding JP is supported by the Else Kröner-Fresenius-Stiftung (2022_EKEA.55). Competing Interests The authors declare no competing interests. Ethical approval Charité ethics approval number: EA2/087/15. Data availability The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. References Kaschta D et al. Evaluating genome sequencing strategies: trio, singleton, and standard testing in rare disease diagnosis. Genome Med 2025; 17: 100. Figueroa KP et al. A GGC-repeat expansion in ZFHX3 encoding polyglycine causes spinocerebellar ataxia type 4 and impairs autophagy. Nat Genet 2024; 56: 1080–1089. Pozojevic J et al. LINE1-mediated epigenetic repression of androgen receptor transcription causes androgen insensitivity syndrome. Sci Rep 2024; 14: 16302. Poretti A et al. Delineation and diagnostic criteria of Oral-Facial-Digital Syndrome type VI. Orphanet J Rare Dis 2012; 7: 4. Qian W et al. Whole-exome sequencing identified novel variants in CPLANE1 that causes oral-facial-digital syndrome Ⅵ by inducing primary cilia abnormality. J Cell Mol Med 2022; 26: 3213–3222. Lopez E et al. C5orf42 is the major gene responsible for OFD syndrome type VI. Hum Genet 2014; 133: 367–377. Srour M et al. Mutations in C5ORF42 cause Joubert syndrome in the French Canadian population. Am J Hum Genet 2012; 90: 693–700. Richards S et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015; 17: 405–424. Abou Tayoun AN et al. Recommendations for interpreting the loss of function PVS1 ACMG/AMP variant criterion. Hum Mutat 2018; 39: 1517–1524. Konkel MK et al. Sequence Analysis and Characterization of Active Human Alu Subfamilies Based on the 1000 Genomes Pilot Project. Genome Biol Evol 2015; 7: 2608–2622. Hancks DC, Kazazian HH Jr. Roles for retrotransposon insertions in human disease. Mob DNA 2016; 7: 9. Payer LM, Burns KH. Transposable elements in human genetic disease. Nat Rev Genet 2019; 20: 760–772. Elbarbary RA, Lucas BA, Maquat LE. Retrotransposons as regulators of gene expression. Science 2016; 351: aac7247. Du AY, Chobirko JD, Zhuo X, Feschotte C, Wang T. Regulatory transposable elements in the encyclopedia of DNA elements. Nat Commun 2024; 15: 7594. Sreenivasan VKA, Yumiceba V, Spielmann M. Structural variants in the 3D genome as drivers of disease. Nat Rev Genet 2025; 26: 742–760. Additional Declarations There is no duality of interest Cite Share Download PDF Status: Under Review Version 1 posted Reviewer # 2 agreed at journal 12 May, 2026 Review # 1 received at journal 07 Apr, 2026 Reviewer # 1 agreed at journal 07 Apr, 2026 Reviewers invited by journal 18 Mar, 2026 Submission checks completed at journal 12 Mar, 2026 First submitted to journal 12 Mar, 2026 Unknown event 11 Mar, 2026 Editor assigned by journal 11 Mar, 2026 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-9094779","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Brief Communication","associatedPublications":[],"authors":[{"id":608203519,"identity":"0154a7c8-a1c9-40ee-966e-03d31b67cbcf","order_by":0,"name":"Malte Spielmann","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIiWNgGAWjYFACxgaGByCavYEBwgADNgJaEkA0zwEGCIOwFgaoSokEIrXIz25ufJCYY5dnLvk68UHCL5s8BrGzh18wlNng1GJw52CzQeK25GLL2bmbDRL70ooZpPPSLBjOpeHWIpHYJpG4jTlxw+3cbRKJPYcTG6RzzAwY2w7jdtiMxPYfidvqEzfcPAvS8h+m5T9uz9xIbGNI3HY4ccMN3m0SCT8OgLQYP2BsO4DXL0CHHU/ccAbkl4bkxDagXxgSziXjdtjs9ocfPm6rTtxw/OzGBx/+2CX2S+ce/vChzA63wySQOYxtoBjhYQPFEZFaGP6ACB7mD/h0jIJRMApGwYgDAN6eX+dRfhK4AAAAAElFTkSuQmCC","orcid":"","institution":"Max Planck Institute for Molecular Genetics","correspondingAuthor":true,"prefix":"","firstName":"Malte","middleName":"","lastName":"Spielmann","suffix":""},{"id":608203520,"identity":"7ca4c1b2-82a0-4448-90f5-07083a5aba42","order_by":1,"name":"Jelena Pozojevic","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jelena","middleName":"","lastName":"Pozojevic","suffix":""},{"id":608203521,"identity":"ec4818ac-79bc-4e51-819f-cf2a61efc571","order_by":2,"name":"Henrike Sczakiel","email":"","orcid":"","institution":"Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin","correspondingAuthor":false,"prefix":"","firstName":"Henrike","middleName":"","lastName":"Sczakiel","suffix":""},{"id":608203522,"identity":"deb67f8c-f0cb-4320-b706-926069011986","order_by":3,"name":"Saranya Balachandran","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Saranya","middleName":"","lastName":"Balachandran","suffix":""},{"id":608203523,"identity":"591720d8-e40a-4d10-be18-96448a835d4d","order_by":4,"name":"Nathalie Kruse","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Nathalie","middleName":"","lastName":"Kruse","suffix":""},{"id":608203524,"identity":"d2585f54-41c4-481d-a2c2-bb4d620ebee3","order_by":5,"name":"Martin Mensah","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"","lastName":"Mensah","suffix":""},{"id":608203525,"identity":"fd0a7aef-efe9-454a-83dd-bea357fdc60b","order_by":6,"name":"Wiebke Hülsemann","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Wiebke","middleName":"","lastName":"Hülsemann","suffix":""},{"id":608203526,"identity":"a47bd96b-46cc-46bf-a5c6-e0ca57b01ae4","order_by":7,"name":"Kristian Händler","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kristian","middleName":"","lastName":"Händler","suffix":""}],"badges":[],"createdAt":"2026-03-11 13:11:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9094779/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9094779/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105195556,"identity":"4826153d-1a7b-46da-b0bb-593c1efc1206","added_by":"auto","created_at":"2026-03-23 10:13:20","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1111945,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotype and genotype of the patient.\u003c/strong\u003e A X-rays of the left hand showing ulnar ray duplication with rudimentary duplication of the ulnar finger and right foot showing duplicated 1st ray with additional duplication of the fibular 1st ray at metatarsophalangeal level \u0026nbsp;with kissing bracket physes of the proximal phalanx; duplication of the 5th toe. B Phased reads showing the biallelic variants c.8633-4_8633-3del in intron 44, and an Alu element insertion at the exon14/ intron 13 boundary, together with the target site duplication (highlighted in yellow) and the entire AluYa5 sequence.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9094779/v1/7e35810396fc23e373cee646.jpg"},{"id":105195455,"identity":"0f846e91-34f1-4720-8635-2636c38cd79f","added_by":"auto","created_at":"2026-03-23 10:13:14","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":493874,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathogenic splicing potential of the two variants in the patient-derived fibroblasts.\u003c/strong\u003e A Variant c.8633-4_8633-3del leads to exon 45 skipping. B AluYa5 transposable element insertion leads to exon 14 skipping. Sequences of both wild type and mutant allele are depicted above the respective electropherograms. C Expression analysis of \u003cem\u003eCPLANE1\u003c/em\u003e mRNA levels in fibroblasts of the index patient compared with three healthy controls. Expression levels were normalized relative to \u003cem\u003eGAPDH\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9094779/v1/898208cd3e132e4b5ba48068.jpg"},{"id":105195459,"identity":"4952a65e-8f2f-462f-bf22-9f59c6a5c4f7","added_by":"auto","created_at":"2026-03-23 10:13:14","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":97372,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVisualization of the Alu insertion in short-read WGS. \u003c/strong\u003eIGV visualization of short-read WGS data at the Alu insertion locus. Multiple reads display soft-clipped alignments at the breakpoint, indicating the presence of an insertion that was not detected by standard variant calling pipelines.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9094779/v1/37ae46806883ee1f8562ddd3.jpg"},{"id":105564129,"identity":"f826d3c8-8e89-46d0-bef5-e44f13e3f22a","added_by":"auto","created_at":"2026-03-27 12:48:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2107033,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9094779/v1/cf8f3ead-0a88-4edc-a209-f2dbbb6dc37a.pdf"}],"financialInterests":"There is no duality of interest","formattedTitle":"Long-read sequencing reveals a hidden Alu-mediated splice defect in CPLANE1, causing orofaciodigital syndrome type VI","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNext-generation sequencing (NGS) has transformed molecular diagnostics, and whole exome sequencing (WES) is widely used to detect protein-coding, disease-causing variants. However, whole genome sequencing (WGS) has the potential to replace multiple diagnostic methods such as chromosome analysis, array-CGH and WES by providing a single diagnostic test\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Despite their high accuracy, short-read sequencing technologies struggle to resolve complex genomic regions, including repetitive and GC-rich sequences. Long-read sequencing overcomes these limitations, enabling improved detections of structural variants, repeat expansions, and epigenetic modifications, as we recently showed on examples of the GGC-repeat expansion in \u003cem\u003eZFHX3\u003c/em\u003e and changed DNA methylation due to a retrotransposon insertion\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOrofaciodigital syndrome type VI (OFD VI) (OMIM #277170) is an autosomal recessive ciliopathy that belongs to the Joubert syndrome related disorders. Diagnostic criteria for OFD VI include the molar tooth sign and one or more of the following: 1) tongue hamartoma(s) and/or additional frenula and/or upper lip notch; 2) mesoaxial polydactyly of one or more hands or feet; 3) hypothalamic hamartoma\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The primary gene responsible for OFD VI is \u003cem\u003eCPLANE1\u003c/em\u003e, (also called C5orf42), which encodes a protein involved in ciliogenesis, cell migration, and Hedgehog signaling\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Pathogenic variants in this gene are also linked to Joubert syndrome 17 (OMIM #614615)\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we present a patient initially diagnosed with excessive polydactyly, cleft lip, and abnormal tongue, with a family history of polydactyly. While short-read WGS failed to identify the genetic cause of the disease, long-read WGS revealed two biallelic splice-site variants in \u003cem\u003eCPLANE1\u003c/em\u003e. Further functional assays on patient-derived fibroblasts revealed pathogenic aberrant splicing effects and decreased \u003cem\u003eCPLANE1\u003c/em\u003e. Our work emphasizes the utility of long-read sequencing especially in detecting clinically relevant transposable element insertions, and re-classifies a rare splice-site variant present in the general population.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eCell culture and DNA extraction\u003c/p\u003e \u003cp\u003eFollowing informed consent, patient-derived fibroblasts were obtained from a skin biopsy and cultured under standard conditions. High molecular weight DNA was extracted using MagAttract HMW DNA kit (Qiagen), according to the manufacturer's protocol.\u003c/p\u003e \u003cp\u003eLong-read sequencing and data analysis\u003c/p\u003e \u003cp\u003ePacBio HiFi long-read sequencing was performed on sheared genomic DNA. Libraries were size-selected to exclude fragments shorter than 10 kb and sequenced on a Sequel II platform. Reads were aligned to the GRCh38 reference genome, followed by variant calling and phasing. Variant interpretation was conducted using the Geneyx software, and visualization was performed with the integrated genomic viewer.\u003c/p\u003e \u003cp\u003eRNA extraction and quantitative PCR (qPCR)\u003c/p\u003e \u003cp\u003eRNA was extracted from patient-derived and control fibroblasts and reverse-transcribed into cDNA. Quantitative PCR (qPCR) was used to assess \u003cem\u003eCPLANE1\u003c/em\u003e expression levels relative to controls. Due to the single-patient design, results were evaluated descriptively without statistical testing.\u003c/p\u003e \u003cp\u003eSanger sequencing\u003c/p\u003e \u003cp\u003eTargeted PCR amplification and Sanger sequencing were performed to validate identified variants and assess their effects on mRNA splicing.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eClinical report\u003c/p\u003e \u003cp\u003eAt 1.5 years of age the male patient presented with postaxial polydactyly of both hands, polydactyly of the left foot, and polysyndactyly of the right foot (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The proband is the first son of healthy, non-consanguineous parents. Family history was positive for polydactyly, with father's cousin (child of consanguineous parents) also reported to show polydactyly. Additionally, the patient presented with a cleft lip and an abnormally shaped tongue. A follow-up evaluation at the age of 6 years additionally revealed global developmental delay (walking independently at the age of 3.5 years, still non-verbal at the current age of 6 years), muscular hypotonia, myopia and hearing impairment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMolecular genetics findings\u003c/p\u003e \u003cp\u003eShort-read WGS did not detect the disease-causing variant(s) and thus the patient was selected for long-read WGS (LR-WGS). Long-read PacBio HiFi sequencing revealed two \u003cem\u003eCPLANE1\u003c/em\u003e variants as the most plausible cause of the phenotype: c.8633-4_8633-3del (NM_001384732.1) and an Alu element insertion at the exon 14/ intron 13 boundary. Phasing the reads showed that they are located on two alleles, consistent with recessive disease inheritance mode (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The c.8633-4_8633-3del is present in gnomAD v4.1 at allele frequency 0.00002653 and classified as a variant of unknown significance (VUS) both in gnomAD and ClinVar. In contrast, the Alu element insertion is not present in gnomAD, and the sequence analysis revealed that it belongs to the AluYa5 family, a young and highly active group of Alu elements. This Alu insertion is flanked by the target site duplication (TSD) sequence, a hallmark of retrotransposon insertions. Based on the TSD sequence, the genomic location of this Alu element insertion is left-normalized and defined to be chr5:37,224,330 (GRCh38).\u003c/p\u003e \u003cp\u003eFunctional assessments of the variants\u003c/p\u003e \u003cp\u003eSince both these variants are intronic, close to the exon-intron boundaries, we next tested their pathogenic splicing potential. Sanger sequencing of mRNA extracted from patient-derived fibroblasts revealed that each of these variants leads to exon skipping: c.8633-4_8633-3del leads to exon 45 skipping (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), while the AluYa5 insertion leads to exon 14 skipping (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Both events result in frameshift (exon 45 is 81 bp long, while exon 14 is 31 bp long), predicted to trigger nonsense-mediated mRNA decay (NMD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eQuantitative PCR confirmed a substantial reduction of \u003cem\u003eCPLANE1\u003c/em\u003e mRNA levels in the patient compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), supporting biallelic loss-of-function.\u003c/p\u003e \u003cp\u003eRe-analysis of the short-read WGS data did not identify the Alu insertion in either structural variant or single nucleotide variant call sets. However, subsequent inspection of the corresponding BAM files revealed soft-clipped reads at the insertion site (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur patient was initially diagnosed with polydactyly, with positive family history, but received no molecular diagnosis even after WGS using short-read technologies. Because of this, the patient was selected for long-read WGS, which indeed detected a previously missed second hit in a recessive gene, an Alu element insertion. Phasing the long reads enabled us to show biallelic nature of the two \u003cem\u003eCPLANE1\u003c/em\u003e variants, while further functional validation demonstrated their pathogenic potential, resulting in loss-of-function. Our work demonstrated that the c.8633-4_8633-3del in intron 44, ultra rare in the general population, leads to exon 45 skipping and frameshift, allowing us to re-classify it as pathogenic (instead of VUS), according to the American College of Medical Genetics and Genomics (ACMG) criteria\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Similarly, the Alu element insertion close to the exon-intron boundary, absent in population databases, leads to exon 14 skipping and frameshift, and we classify this variant as pathogenic based on the ACMG criteria (PVS1, PS3, PM2, PM3). Further functional evidence that demonstrates pathogenicity of both variants includes markedly decreased \u003cem\u003eCPLANE1\u003c/em\u003e transcript levels in patient-derived fibroblasts, as detected by qPCR, consistent with frameshift variants predicted to undergo NMD.\u003c/p\u003e \u003cp\u003eOf note, the molecular diagnosis by LR-WGS was based solely on the information that the patient presents with polydactyly, and the variant interpretation was based on the human phenotype ontology terms (HP:0010442, HP:0010689). Revisiting the phenotype at the age of 6 years showed that the patient presents with neurodevelopmental delay in addition to the initially observed polydactyly, which led the clinician to suspect OFD syndrome. Thus, the molecular diagnosis is in accordance with the clinical diagnosis, establishing that the patient presents with OFD VI. The Alu insertion was not detected by standard variant calling approaches applied to short-read WGS data, highlighting a limitation of this methodology for identifying transposable element insertions. Its detection by PacBio long-read sequencing underscores the added value of long-read technologies in resolving such variants. Retrospective examination of the short-read alignments revealed soft-clipped reads at the locus, suggesting that while signals of the insertion were present, they were not captured by conventional variant calling pipelines.\u003c/p\u003e \u003cp\u003eTransposable elements (TEs) constitute nearly 50% of the human genome. While many of them are evolutionary ancient and inactive, several families remain active and capable of mobilization, most notably Alu, LINE, and SVA elements. As a result, they contribute both to phenotypic diversity and human disease. Despite their biological and clinical relevance, TEs are frequently underdetected by conventional sequencing approaches, including short-read technologies. This limitation arises in part from their highly repetitive nature and widespread genomic distribution, which complicates accurate read mapping and insertion site resolution. Additionally, many TEs are GC-rich, leading to amplification bias and reduced coverage due to inefficient polymerase activity during amplification. Consequently, the full extent of their variation and activity remains incompletely characterized using standard sequencing methods. AluYa5 family is the youngest and most active among Alu elements, and has been associated with human disease, including neurodevelopmental disorders and cancer\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Insertions within introns or regulatory regions can disrupt gene expression through altered splicing, premature polyadenylation, or transcriptional interference, depending on the location of their insertion and the genomic context\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. It is tempting to speculate whether the Alu element identified in our patient is a \u003cem\u003ede novo\u003c/em\u003e insertion or is absent in population databases due to limitations of short-read sequencing technologies. In conclusion, as LR-WGS enters clinical diagnostics, our work highlights its clear technical advantages in mapping accuracy, sequence resolution, and variant phasing \u0026mdash; without requiring parental samples or additional downstream analyses. Beyond this, LR-WGS offers added strengths, including integrated epigenetic profiling and the potential to function as a single, comprehensive diagnostic test. Moreover, LR-WGS promises to provide deeper insight into the non-coding genome, enabling the identification of previously undetected regulatory variants and disease mechanisms\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to the patient and his family for participating in this study.\u003c/p\u003e\n\u003cp\u003eAuthor Contribution Statement\u003c/p\u003e\n\u003cp\u003eJP analyzed LR-WGS data, performed the functional assays, and wrote the first draft of the manuscript. HLS performed clinical assessment of the patient and edited the manuscript. NK prepared the sample for LR-WGS. SB performed bioinformatic analyses. MAM performed clinical assessment of the patient. WH performed surgery and took the skin specimen. KH supervised the LR-WGS and edited the manuscript. MS supervised the entire project and edited the manuscript.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eJP is supported by the Else Kr\u0026ouml;ner-Fresenius-Stiftung (2022_EKEA.55).\u003c/p\u003e\n\u003cp\u003eCompeting Interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eEthical approval\u003c/p\u003e\n\u003cp\u003eCharit\u0026eacute; ethics approval number: EA2/087/15.\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKaschta D \u003cem\u003eet al.\u003c/em\u003e Evaluating genome sequencing strategies: trio, singleton, and standard testing in rare disease diagnosis. \u003cem\u003eGenome Med\u003c/em\u003e 2025; 17: 100.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFigueroa KP \u003cem\u003eet al.\u003c/em\u003e A GGC-repeat expansion in ZFHX3 encoding polyglycine causes spinocerebellar ataxia type 4 and impairs autophagy. \u003cem\u003eNat Genet\u003c/em\u003e 2024; 56: 1080\u0026ndash;1089.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePozojevic J \u003cem\u003eet al.\u003c/em\u003e LINE1-mediated epigenetic repression of androgen receptor transcription causes androgen insensitivity syndrome. \u003cem\u003eSci Rep\u003c/em\u003e 2024; 14: 16302.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoretti A \u003cem\u003eet al.\u003c/em\u003e Delineation and diagnostic criteria of Oral-Facial-Digital Syndrome type VI. \u003cem\u003eOrphanet J Rare Dis\u003c/em\u003e 2012; 7: 4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQian W \u003cem\u003eet al.\u003c/em\u003e Whole-exome sequencing identified novel variants in CPLANE1 that causes oral-facial-digital syndrome Ⅵ by inducing primary cilia abnormality. \u003cem\u003eJ Cell Mol Med\u003c/em\u003e 2022; 26: 3213\u0026ndash;3222.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLopez E \u003cem\u003eet al.\u003c/em\u003e C5orf42 is the major gene responsible for OFD syndrome type VI. \u003cem\u003eHum Genet\u003c/em\u003e 2014; 133: 367\u0026ndash;377.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSrour M \u003cem\u003eet al.\u003c/em\u003e Mutations in C5ORF42 cause Joubert syndrome in the French Canadian population. \u003cem\u003eAm J Hum Genet\u003c/em\u003e 2012; 90: 693\u0026ndash;700.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRichards S \u003cem\u003eet al.\u003c/em\u003e Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. \u003cem\u003eGenet Med\u003c/em\u003e 2015; 17: 405\u0026ndash;424.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbou Tayoun AN \u003cem\u003eet al.\u003c/em\u003e Recommendations for interpreting the loss of function PVS1 ACMG/AMP variant criterion. \u003cem\u003eHum Mutat\u003c/em\u003e 2018; 39: 1517\u0026ndash;1524.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKonkel MK \u003cem\u003eet al.\u003c/em\u003e Sequence Analysis and Characterization of Active Human Alu Subfamilies Based on the 1000 Genomes Pilot Project. \u003cem\u003eGenome Biol Evol\u003c/em\u003e 2015; 7: 2608\u0026ndash;2622.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHancks DC, Kazazian HH Jr. Roles for retrotransposon insertions in human disease. \u003cem\u003eMob DNA\u003c/em\u003e 2016; 7: 9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePayer LM, Burns KH. Transposable elements in human genetic disease. \u003cem\u003eNat Rev Genet\u003c/em\u003e 2019; 20: 760\u0026ndash;772.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElbarbary RA, Lucas BA, Maquat LE. Retrotransposons as regulators of gene expression. \u003cem\u003eScience\u003c/em\u003e 2016; 351: aac7247.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDu AY, Chobirko JD, Zhuo X, Feschotte C, Wang T. Regulatory transposable elements in the encyclopedia of DNA elements. \u003cem\u003eNat Commun\u003c/em\u003e 2024; 15: 7594.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSreenivasan VKA, Yumiceba V, Spielmann M. Structural variants in the 3D genome as drivers of disease. \u003cem\u003eNat Rev Genet\u003c/em\u003e 2025; 26: 742\u0026ndash;760.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-human-genetics","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ejhg","sideBox":"Learn more about [European Journal of Human Genetics](http://www.nature.com/ejhg/)","snPcode":"41431","submissionUrl":"https://mts-ejhg.nature.com/cgi-bin/main.plex","title":"European Journal of Human Genetics","twitterHandle":"@ejhg_journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9094779/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9094779/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOrofaciodigital syndrome type VI (OFD VI) is a recessive ciliopathy characterized by excessive polydactyly, molar tooth sign, cleft lip, and developmental delay, caused by pathogenic variants in \u003cem\u003eCPLANE1\u003c/em\u003e. Here, we present a patient with OFD VI that remained genetically unexplained after routine genetic testing, including short-read whole genome sequencing (WGS). Using long-read sequencing, we found two biallelic splice-site variants in \u003cem\u003eCPLANE1\u003c/em\u003e, c.8633-4_8633-3del, and an Alu element insertion close to an exon-intron boundary. Transcript analysis showed that each variant independently resulted in exon skipping, and quantitative expression studies revealed reduced total \u003cem\u003eCPLANE1\u003c/em\u003e mRNA levels in patient-derived fibroblasts. Based on these findings, we were able to re-classify the c.8633-4_8633-3del variant from a variant of uncertain significance (VUS) to pathogenic. The identification of an Alu element insertion missed by short-read WGS highlights the added diagnostic value of long-read sequencing in uncovering cryptic, transposable element-associated pathogenic variants.\u003c/p\u003e","manuscriptTitle":"Long-read sequencing reveals a hidden Alu-mediated splice defect in CPLANE1, causing orofaciodigital syndrome type VI","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-23 10:12:14","doi":"10.21203/rs.3.rs-9094779/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-12T21:37:12+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-04-07T21:36:01+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-04-07T20:43:38+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-03-18T10:59:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-12T09:53:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Human Genetics","date":"2026-03-12T07:51:31+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2026-03-11T15:46:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-11T12:57:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-human-genetics","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ejhg","sideBox":"Learn more about [European Journal of Human Genetics](http://www.nature.com/ejhg/)","snPcode":"41431","submissionUrl":"https://mts-ejhg.nature.com/cgi-bin/main.plex","title":"European Journal of Human Genetics","twitterHandle":"@ejhg_journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7888b8df-4e0e-4bc7-9c15-4b51255b632b","owner":[],"postedDate":"March 23rd, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-12T21:37:12+00:00","index":2,"fulltext":"This content is not available."}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":64719069,"name":"Health sciences/Medical research/Genetics research"},{"id":64719070,"name":"Biological sciences/Genetics/Gene expression"}],"tags":[],"updatedAt":"2026-03-23T10:12:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-23 10:12:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9094779","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9094779","identity":"rs-9094779","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}