Combined Optical Genome Mapping and CNV-Seq indentify Complex Y-Chromosome Rearrangements and Ectopy in 46,XX Testicular Disorder of Sex Development

Combined Optical Genome Mapping and CNV-Seq indentify Complex Y-Chromosome Rearrangements and Ectopy in 46,XX Testicular Disorder of Sex Development | 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 Research Article Combined Optical Genome Mapping and CNV-Seq indentify Complex Y-Chromosome Rearrangements and Ectopy in 46,XX Testicular Disorder of Sex Development Hai Wang, Hao Wang, Zitong Xu, Xianjue Zheng, Haojie Pan, Hongping Zhang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7475133/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective To clarify the chromosomal structural variations in a patient with 46,XX testicular disorder of sex development (DSD) using combined genetic technologies. Methods Chromosome karyotype analysis and whole exome sequencing (WES) were initially performed in a phenotypically male patient. Further analyses using optical genome mapping (OGM) and CNV-seq were conducted on fresh peripheral blood to characterize structural variations. Result Karyotyping revealed 46,XX,inv(9)(p12q13), and WES suggested possible Y-chromosome sequences. CNV-seq indicated deletions in Xp22.33, Yp11.31-p11.2, and Yp11.2, confirming 46,XX testicular DSD. OGM futher demonstrated a structural translocation Yp segments to the X chromosome short arm, with three breakage-reconnection events in Yp11.2, including deletions and inversions, clarifying the complex Y-chromosome rearrangements and derivative X-chromosome structure. Conclusion Combined OGM and CNV-seq precisely localized Y-chromosome ectopy to the X short arm and characterized its complex rearrangements, providing patients with more accurate and comprehensive genetic diagnosis, and which has important clinical significance for genetic counseling of sex reversal syndrome. Sex Reversal Syndrome Structural Variation OGM CNV-seq Y-chromosome Translocation Figures Figure 1 Figure 2 Figure 3 Introduction Sex reversal syndrome (SRS) is a condition in which a person's chromosome karyotype does not match his or her phenotypic sex, either as a 46,XX male or as a 46,XY female. Among them, 46,XX Testicular Disorder of Sex Development (DSD)is caused by SRY gene translocation or SOX9 sex-determining pathway abnormality 1 , 2 . The phenotype of this kind of patients is mostly male, with the characteristics of testicular dysplasia, low testosterone level, hypospadias, significantly increased gonadotropin level, lower height than normal male, azoospermia, underdeveloped secondary sexual characteristics, small Adam's apple, fine skin and different degrees of male breast development. The incidence of this disease in neonatal males is 1/20,000 3–6 . Optical Genome Mapping (OGM) is a new high-resolution cytogenetic analysis method and is considered as the next generation cytogenetic technology. By combining microfluidics, automatic image analysis and high-resolution genome sequencing technologies, OGM can rapidly generate genome-wide high-resolution restriction enzyme maps with extremely high sensitivity and accuracy. OGM can detect almost all clinically significant structural variants (SV) in samples. These include aneuploids, copy number variations (CNVs) such as deletions and duplications, balanced and unbalanced translocations, inversions, insertions, circular chromosomes, and loss of heterozygosity 7 , 8 . OGM and CNV-seq techniques were used to analyze a male child with chromosome 46,XX born in hospital. The study reveals the critical role of OGM and CNV-seq technologies in the diagnosis of Disorders of Sex Development (DSD), specifically in cases of sex reversal syndrome. Materials and Methods Participants The child is one and a half years old, and his appearance is male, unfused scrotum, hypospadias after birth. Dehydroepiandrosterone sulfate 104ng/ml, aldosterone (supine) 48.6 ng/dl, plasma renin (supine) 48.92 uIU/ml. Chromosome karyotype analysis and whole exon gene sequencing (WES) were performed at the same time to check the possibility of chromosome and gene disease. The results showed that chromosome karyotype was 46, XX, inv (9)(p12q13). WES did not detect any variation related to the phenotype of the child, but its supplementary report suggested 47,XXY,+X[seq(GRCh37/hg19)].; (X)×2 indicates the presence of Y chromosome. His father is 32 years old and has a normal male karyotype, and his mother is 29 years old and has a normal female karyotype. The mother's prenatal NIPT-PLUS results were negative, and the relevant prenatal ultrasound examinations were normal. In order to clarify the specific variation mode of Y chromosome, further investigations were performed on this basis. Collection of specimens Fresh EDTA anticoagulant peripheral blood was collected for OGM and CNV-seq detection. OGM test The OGM analysis platform provided by Bionano Genomics (San Diego, CA, USA) was used for OGM detection. The detection process was as follows: (1) Extraction of ultra-long DNA. Bionano Prep SP Blood and Cell DNA Isolation kit v2 (Part 80042) was used to extract sample DNA and obtain DNA fragments at megabase level by adsorption disk technology. The key is to obtain ultra-long DNA containing SV information. (2) DNA-specific fluorescent labeling and staining. High molecular weight DNA was labeled and stained by direct labeling staining technique. The labeling enzyme recognized CTTAAG sequence and added green fluorescence, followed by staining with blue dye to form blue DNA molecules with green fluorescence signal. (3) Single molecule DNA linearization. Labeled gDNA was loaded onto the Saphyr chip and linearized in micro and nano channels by low voltage electric fields. (4) High-resolution imaging of single-molecule DNA. The high-resolution fluorescence microscope of the Saphyr instrument captures single-molecule DNA, and the generated original images are uploaded to the Bionano platform and converted into molecular files containing the molecular length and CTTAAG position. (5) Genome assembly and alignment. The analysis software assembles the genomic map using the CTTAAG position information, compares it with the reference sequence, and conducts structural variation analysis, including copy number changes and genomic rearrangements. OGM also detects CNV and aneuploidy through the CNV algorithm. (6) Structural Variation report. After the assembly of the optical atlas is completed, data processing and analysis are carried out using Bionano Solve and Access software to generate a structural variation report. CNV-seq test The patient's blood samples were sent to Shenzhen Huada Medical Laboratory for chromosome copy number variation testing. The specific process includes: extracting genomic DNA with nucleic acid extraction kit, constructing library after enzyme digestion, end repair and adapter ligation, and sequencing single-end 35bp (average depth 0.4×) by DNBSEQ-T7 sequencing platform (combined probe-anchored polymerization sequencing method). After GRCh37 reference genome alignment, GC correction and de-duplication processing, CNV analysis (resolution ≥ 100 kb) was performed using Tattini algorithm, and pathogenicity annotation was performed in combination with OMIM ( https://www.deciphergenomics.org/ ), DECIPHER ( https://www.ncbi.nlm.nih.gov/projects/dbvar/clingen/index.shtml ) databases. Finally, the pathogenicity of CNV was graded according to the American College of Medical Genetics and Genomics (ACMG) standard. Result OGM test results OGM test results are: ogm[GRCh38] der(X)(Ypter_Yp11.2::Yp11.2::Xp22.33_Xqter)(Ypter_6248448::9915169_7362950::3618476_Xqter), it is speculated that the derivative X chromosome detected this time is caused by partial region translocation from the end of Y chromosome short arm to the end of X chromosome short arm, and Y chromosome has three breakage-reconnection events in Yp11.2 region, among which Yp11.2 6.2 Mb-7.3 M region is deleted after break, and Yp11.2 7.3 Mb-9.9 Mb region is inverted reconnection after break (Fig. 1 ). CNV-seq test results The results suggested that seq[GRCh37] Xp22.33p22.33(287,328–3,605,487) × 1, Yp11.31p11.2 (2649472–6065425) × 1, Yp11.2p11.2 (7270527–9599178) ×1 (Fig. 2 ). It is suggested that there is a single copy deletion at band 22.33 on the short arm of X chromosome and a single copy deletion at bands 11.2 and 11.31 on the short arm of Y chromosome, which can be diagnosed as 46,XX DSD. Ultrasound test results of children Doppler ultrasound showed testicle echoes in left and right scrotum, no obvious abnormality in bilateral epididymis, no obvious uterus echo behind bladder, no obvious ovary echo in abdominal cavity (Fig. 3 ). Discussion In this study, we reported a patient with 46,XX DSD. The patient was male in appearance. The karyotype of chromosome was 46,XX,inv(9)(p12q13) found by routine peripheral blood karyotype analysis. After WES and CNV-seq examination, it was definitely diagnosed as 46,XX DSD. The subsequent OGM technology clarified the specific site and mode of repeated Y chromosome ectopic. 46,XX testicular DSD is classified into SRY positive and SRY negative according to the presence or absence of SRY gene, among which SRY positive patients accounted for about 80%, most of which were caused by the translocation of SRY gene on Y chromosome to X chromosome during father meiosis 9 – 12 . The mechanism of the disease is that the pseudoautosomal region (PAR) at the end of Y chromosome is homologous to the end of X chromosome 13 , 14 , and SRY gene is very close to PAR. During the process of meiotic homologous chromosome pairing and exchange, the exchange at PAR boundary region can cause SRY gene to be transferred into X chromosome, that is, non-allelic homologous recombination occurs, so that the patient can still have male gonad development without Y chromosome 15 – 17 . This patient is SRY positive, 1 year of age, male genitalia appeared normal by ultrasound examination. Because of the existence of extra X chromosome in the body, it may affect the normal development of testis, lead to testicular dysplasia, testosterone level decrease, and may appear hypogonadism, testicular dysplasia and infertility after puberty 18 . However, due to timely diagnosis, hormone therapy can be used to maintain the normal development of secondary sexual characteristics and quality of life 19 , 20 . According to the literature review, more than one hundred cases of 46,XX DSD have been reported in the medical literature 9 , 21 . Diagnostic work for 46,XX DSD includes clinical phenotyping, endocrine assessment, karyotype analysis, and molecular detection of Y-derived sequences such as SRY by fluorescence quantitative polymerase chain reaction (QF-PCR) and fluorescence in situ hybridization (FISH). However, even if the above technologies are combined, there are still many limitations. These techniques have a limited range of applications and can only detect the presence of SRY genes on the Y chromosome and roughly determine the location of the Y chromosome. In addition, cross-use is not only time-consuming, but also requires multiple tests. CNV-seq and OGM have been used to detect Y chromosome variation and complex Y chromosome rearrangement. CNV-seq detects the location and size of copy number variation by extracting sample DNA and cutting it into short fragments, then performing high-throughput sequencing, generating a large number of read data, analyzing these data using bioinformatics algorithms, and calculating read count differences within sliding windows 22 . CNV-seq can detect copy number variation as low as 100 kb, but cannot identify balanced SVs and insertions and deletions < 50 kb 23 , 24 . By directly imaging fluorescently labeled ultra-high molecular weight (UHMW) DNA, OGM can accurately and automatically identify clinically significant balanced and unbalanced SV in clinical samples. At present, many scientific experiments and clinical confirmatory studies have proved that, as a new generation of cell genomics technology, OGM can detect all kinds of balanced and unbalanced SV and numerical abnormalities as low as 0.5kb at one time, with high resolution 25 ,26 and 100% coincidence rate with clinical standard of care (SOC) in sensitivity, specificity, positive predictive value and negative predictive value 27 . However, the same OGM has insufficient detection performance for heterochromatin region 28 . The combination of the two techniques in this study improved the sensitivity and accuracy of the test results. CNV-seq technology is based on second-generation sequencing and has low cost, but its resolution ability for complex regions (such as repetitive sequences or GC-rich regions) is limited 29 , and its detection ability for PAR regions is insufficient 30 . OGM technology can accurately resolve structural variations and repetitive sequences in PAR regions. The reliability of the results was cross-verified by the two technologies. Agnethe et al. 31 detected the Y chromosome of 11 patients with 46,XX DSD by long-read sequencing technique, among which 1 patient did not detect SRY gene and other genetic causes leading to male sexual development, and the other 10 patients detected the breakpoints of Y chromosome and X chromosome, and the smallest fragment size was 2,782 kb. Therefore, based on the characteristics of OGM technology, the diagnostic ability of 46,XX DSD patients is similar to that of long-read sequencing technology, and it is also impossible to find SRY-independent pathogenic gene variants such as SOX9 and RSPO1 32–34 . Existing studies have shown that CNV-seq and OGM technologies have their own advantages in 46,XX DSD diagnosis. The former can detect large-segment copy number variations while the latter is good at analyzing fine structural variations. However, both of them have limitations in detecting heterochromatin regions and cannot identify non-SRY dependent pathogenic mutations. In general, the combination of OGM and CNV-seq can detect structural variation patterns with high resolution, thus making more accurate and comprehensive cytogenetic diagnosis, which has clinical significance in genetic counseling. Conclusion 46,XX testicular DSD is a rare disorder with discrepancies between genetic, gonadal, and phenotypic sex. In this case, CNV-seq identified critical deletions in X and Y chromosomes, while OGM further resolved the complex Y-chromosome rearrangements, including translocations, deletions, and inversions, and precisely localized Y-chromosome ectopy to the X short arm. This study highlights that the combination of OGM and CNV-seq overcomes the limitations of traditional diagnostics, enabling high-resolution characterization of structural variations in 46,XX testicular DSD. Such an integrated approach enhances the accuracy of genetic diagnosis, clarifies the pathogenic mechanism, and provides valuable insights for clinical management and genetic counseling in sex reversal syndrome. Declarations Ethical Approval Approval for this study was obtained from the Ethics Review Committee of Wenzhou People’s Hospital (Approval No.KY-202508-035). Competing Interests No competing interest between the authors. Funding: This study was funded by Wenzhou City Major Scientific and Technological innovation Project (ZY2024023), and Basic Research Project of Wenzhou City (Y2023528). Author Contribution HW and ZX drafted the manuscript. XZ and HP conducted the experiments and data analysis. JZ designed and supervised the study. All authors read and approved the final manuscript. Acknowledgements We would like to thank the patient and his family for their participation and cooperation in this study. 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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-7475133","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":509387333,"identity":"3b924445-7bdc-47b7-8545-2e14d942acc4","order_by":0,"name":"Hai Wang","email":"","orcid":"","institution":"Wenzhou City People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hai","middleName":"","lastName":"Wang","suffix":""},{"id":509387340,"identity":"6fd71a6c-22e9-4238-a350-77e6ec19f3dc","order_by":1,"name":"Hao Wang","email":"","orcid":"","institution":"Zhejiang University School of Medicine Affiliated Sir Run Run Shaw Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Wang","suffix":""},{"id":509387342,"identity":"280803ec-d8f4-4380-8d15-5f19e2a6f1e9","order_by":2,"name":"Zitong Xu","email":"","orcid":"","institution":"Wenzhou City People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zitong","middleName":"","lastName":"Xu","suffix":""},{"id":509387348,"identity":"5ed19047-c0d4-4e48-b92e-0cedd4c5b463","order_by":3,"name":"Xianjue Zheng","email":"","orcid":"","institution":"Wenzhou City People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xianjue","middleName":"","lastName":"Zheng","suffix":""},{"id":509387349,"identity":"31b3c4f4-b747-4259-87c8-37c9f6f5b625","order_by":4,"name":"Haojie Pan","email":"","orcid":"","institution":"Wenzhou City People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Haojie","middleName":"","lastName":"Pan","suffix":""},{"id":509387353,"identity":"5223e4a0-9da0-45d0-85b9-d0129a67d2c5","order_by":5,"name":"Hongping Zhang","email":"","orcid":"","institution":"Wenzhou City People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hongping","middleName":"","lastName":"Zhang","suffix":""},{"id":509387358,"identity":"23fde56a-786f-483e-aaaa-cc7b9f1d6742","order_by":6,"name":"Jiayong Zheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYBACxmYowwBEfEBiE6eFcQYxWuAApIyZhxgtzO3MzyQ+7qi1N2fvPfzatm1bYgN78zYJhpo7eBzGZiY588xxZsuec2nWuW23Ext4jpVJMBx7hs8vZtK8bcfYDG7kmBnnbgNqkcgxk2BsOIxHC/s3kBYeg/tvzIwtQVrk3xDSwgOypUbC4AaP8WNGsC08BLUUW85sO2BgcCbHjLH3323jNp60YouEY7i1GPYf33jjY1udvcHxM8Yffpy5LdvPfnjjjQ81eLQ0MLBIMDCAFbBJgEkQkYBTAwODPDBqgMmkDsRm/oBH4SgYBaNgFIxgAAB9KVXWKH2QVgAAAABJRU5ErkJggg==","orcid":"","institution":"Wenzhou City People's Hospital","correspondingAuthor":true,"prefix":"","firstName":"Jiayong","middleName":"","lastName":"Zheng","suffix":""}],"badges":[],"createdAt":"2025-08-28 00:38:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7475133/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7475133/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91016440,"identity":"192e5f97-b42d-4701-9b36-c2ea60d26032","added_by":"auto","created_at":"2025-09-10 16:59:08","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1058951,"visible":true,"origin":"","legend":"\u003cp\u003eGenome structure detected by OGM technique. (a) Genome-wide circos plot showing X and Y ectopic;(b) Schematic diagram of derived X chromosome rearrangement;(c) Ref24 is Y chromosome, and the diagram is based on T2T (Telomere-to-telomere) version of the reference sequence displayed Y chromosome structural variation detection results;(d) Ref23 is X chromosome, Ref24 is Y chromosome, the figure is based on T2T (e) The CNV profile showing a deletion of approximately 3.6 Mb in the X chromosome p22.33 region.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7475133/v1/8bebe2ff935cbaf9d5cd2497.jpeg"},{"id":91015937,"identity":"63014843-9b8b-44a4-bd34-595c04fcca91","added_by":"auto","created_at":"2025-09-10 16:51:08","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":675679,"visible":true,"origin":"","legend":"\u003cp\u003eCNV-seq test results for patients. (a) X chromosome copy number variation was more obvious in band 22.33 of short arm. (b) The distribution of copy number variation on Y chromosome, p11.21 band of short arm has significant copy number variation, while the right region is relatively stable.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7475133/v1/a0104c3772aa9cc4ee77273d.jpeg"},{"id":91016437,"identity":"05f0cc50-3023-4999-84f3-b9d061b95aca","added_by":"auto","created_at":"2025-09-10 16:59:08","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":317320,"visible":true,"origin":"","legend":"\u003cp\u003eDoppler ultrasound images. The ultrasound image showed obvious testicle echo at the intersection of white dotted line, and no obvious abnormality in other parts\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7475133/v1/f6d77abdd7831ed6ef453b6a.jpeg"},{"id":91252847,"identity":"27016d5a-9e48-431c-814e-cc7fa8bf8c02","added_by":"auto","created_at":"2025-09-13 17:46:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2523546,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7475133/v1/25ef037f-fa50-4cab-9cb3-1d35005b7c63.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Combined Optical Genome Mapping and CNV-Seq indentify Complex Y-Chromosome Rearrangements and Ectopy in 46,XX Testicular Disorder of Sex Development","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSex reversal syndrome (SRS) is a condition in which a person's chromosome karyotype does not match his or her phenotypic sex, either as a 46,XX male or as a 46,XY female. Among them, 46,XX Testicular Disorder of Sex Development (DSD)is caused by \u003cem\u003eSRY\u003c/em\u003e gene translocation or \u003cem\u003eSOX9\u003c/em\u003e sex-determining pathway abnormality\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The phenotype of this kind of patients is mostly male, with the characteristics of testicular dysplasia, low testosterone level, hypospadias, significantly increased gonadotropin level, lower height than normal male, azoospermia, underdeveloped secondary sexual characteristics, small Adam's apple, fine skin and different degrees of male breast development. The incidence of this disease in neonatal males is 1/20,000\u003csup\u003e3\u0026ndash;6\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOptical Genome Mapping (OGM) is a new high-resolution cytogenetic analysis method and is considered as the next generation cytogenetic technology. By combining microfluidics, automatic image analysis and high-resolution genome sequencing technologies, OGM can rapidly generate genome-wide high-resolution restriction enzyme maps with extremely high sensitivity and accuracy. OGM can detect almost all clinically significant structural variants (SV) in samples. These include aneuploids, copy number variations (CNVs) such as deletions and duplications, balanced and unbalanced translocations, inversions, insertions, circular chromosomes, and loss of heterozygosity\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOGM and CNV-seq techniques were used to analyze a male child with chromosome 46,XX born in hospital. The study reveals the critical role of OGM and CNV-seq technologies in the diagnosis of Disorders of Sex Development (DSD), specifically in cases of sex reversal syndrome.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eParticipants\u003c/h2\u003e\u003cp\u003eThe child is one and a half years old, and his appearance is male, unfused scrotum, hypospadias after birth. Dehydroepiandrosterone sulfate 104ng/ml, aldosterone (supine) 48.6 ng/dl, plasma renin (supine) 48.92 uIU/ml. Chromosome karyotype analysis and whole exon gene sequencing (WES) were performed at the same time to check the possibility of chromosome and gene disease. The results showed that chromosome karyotype was 46, XX, inv (9)(p12q13). WES did not detect any variation related to the phenotype of the child, but its supplementary report suggested 47,XXY,+X[seq(GRCh37/hg19)].; (X)\u0026times;2 indicates the presence of Y chromosome. His father is 32 years old and has a normal male karyotype, and his mother is 29 years old and has a normal female karyotype. The mother's prenatal NIPT-PLUS results were negative, and the relevant prenatal ultrasound examinations were normal. In order to clarify the specific variation mode of Y chromosome, further investigations were performed on this basis.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCollection of specimens\u003c/h3\u003e\n\u003cp\u003eFresh EDTA anticoagulant peripheral blood was collected for OGM and CNV-seq detection.\u003c/p\u003e\n\u003ch3\u003eOGM test\u003c/h3\u003e\n\u003cp\u003eThe OGM analysis platform provided by Bionano Genomics (San Diego, CA, USA) was used for OGM detection. The detection process was as follows: (1) Extraction of ultra-long DNA. Bionano Prep SP Blood and Cell DNA Isolation kit v2 (Part 80042) was used to extract sample DNA and obtain DNA fragments at megabase level by adsorption disk technology. The key is to obtain ultra-long DNA containing SV information. (2) DNA-specific fluorescent labeling and staining. High molecular weight DNA was labeled and stained by direct labeling staining technique. The labeling enzyme recognized CTTAAG sequence and added green fluorescence, followed by staining with blue dye to form blue DNA molecules with green fluorescence signal. (3) Single molecule DNA linearization. Labeled gDNA was loaded onto the Saphyr chip and linearized in micro and nano channels by low voltage electric fields. (4) High-resolution imaging of single-molecule DNA. The high-resolution fluorescence microscope of the Saphyr instrument captures single-molecule DNA, and the generated original images are uploaded to the Bionano platform and converted into molecular files containing the molecular length and CTTAAG position. (5) Genome assembly and alignment. The analysis software assembles the genomic map using the CTTAAG position information, compares it with the reference sequence, and conducts structural variation analysis, including copy number changes and genomic rearrangements. OGM also detects CNV and aneuploidy through the CNV algorithm. (6) Structural Variation report. After the assembly of the optical atlas is completed, data processing and analysis are carried out using Bionano Solve and Access software to generate a structural variation report.\u003c/p\u003e\n\u003ch3\u003eCNV-seq test\u003c/h3\u003e\n\u003cp\u003eThe patient's blood samples were sent to Shenzhen Huada Medical Laboratory for chromosome copy number variation testing. The specific process includes: extracting genomic DNA with nucleic acid extraction kit, constructing library after enzyme digestion, end repair and adapter ligation, and sequencing single-end 35bp (average depth 0.4\u0026times;) by DNBSEQ-T7 sequencing platform (combined probe-anchored polymerization sequencing method). After GRCh37 reference genome alignment, GC correction and de-duplication processing, CNV analysis (resolution\u0026thinsp;\u0026ge;\u0026thinsp;100 kb) was performed using Tattini algorithm, and pathogenicity annotation was performed in combination with OMIM (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.deciphergenomics.org/\u003c/span\u003e\u003cspan address=\"https://www.deciphergenomics.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), DECIPHER (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/projects/dbvar/clingen/index.shtml\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/projects/dbvar/clingen/index.shtml\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) databases. Finally, the pathogenicity of CNV was graded according to the American College of Medical Genetics and Genomics (ACMG) standard.\u003c/p\u003e"},{"header":"Result","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eOGM test results\u003c/h2\u003e\u003cp\u003eOGM test results are: ogm[GRCh38] der(X)(Ypter_Yp11.2::Yp11.2::Xp22.33_Xqter)(Ypter_6248448::9915169_7362950::3618476_Xqter), it is speculated that the derivative X chromosome detected this time is caused by partial region translocation from the end of Y chromosome short arm to the end of X chromosome short arm, and Y chromosome has three breakage-reconnection events in Yp11.2 region, among which Yp11.2 6.2 Mb-7.3 M region is deleted after break, and Yp11.2 7.3 Mb-9.9 Mb region is inverted reconnection after break (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCNV-seq test results\u003c/h3\u003e\n\u003cp\u003eThe results suggested that seq[GRCh37] Xp22.33p22.33(287,328\u0026ndash;3,605,487) \u0026times; 1, Yp11.31p11.2 (2649472\u0026ndash;6065425) \u0026times; 1, Yp11.2p11.2 (7270527\u0026ndash;9599178) \u0026times;1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). It is suggested that there is a single copy deletion at band 22.33 on the short arm of X chromosome and a single copy deletion at bands 11.2 and 11.31 on the short arm of Y chromosome, which can be diagnosed as 46,XX DSD.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eUltrasound test results of children\u003c/h3\u003e\n\u003cp\u003eDoppler ultrasound showed testicle echoes in left and right scrotum, no obvious abnormality in bilateral epididymis, no obvious uterus echo behind bladder, no obvious ovary echo in abdominal cavity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we reported a patient with 46,XX DSD. The patient was male in appearance. The karyotype of chromosome was 46,XX,inv(9)(p12q13) found by routine peripheral blood karyotype analysis. After WES and CNV-seq examination, it was definitely diagnosed as 46,XX DSD. The subsequent OGM technology clarified the specific site and mode of repeated Y chromosome ectopic.\u003c/p\u003e\u003cp\u003e46,XX testicular DSD is classified into SRY positive and SRY negative according to the presence or absence of SRY gene, among which SRY positive patients accounted for about 80%, most of which were caused by the translocation of SRY gene on Y chromosome to X chromosome during father meiosis\u003csup\u003e\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The mechanism of the disease is that the pseudoautosomal region (PAR) at the end of Y chromosome is homologous to the end of X chromosome\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, and SRY gene is very close to PAR. During the process of meiotic homologous chromosome pairing and exchange, the exchange at PAR boundary region can cause SRY gene to be transferred into X chromosome, that is, non-allelic homologous recombination occurs, so that the patient can still have male gonad development without Y chromosome\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. This patient is SRY positive, 1 year of age, male genitalia appeared normal by ultrasound examination. Because of the existence of extra X chromosome in the body, it may affect the normal development of testis, lead to testicular dysplasia, testosterone level decrease, and may appear hypogonadism, testicular dysplasia and infertility after puberty\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, due to timely diagnosis, hormone therapy can be used to maintain the normal development of secondary sexual characteristics and quality of life\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAccording to the literature review, more than one hundred cases of 46,XX DSD have been reported in the medical literature\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Diagnostic work for 46,XX DSD includes clinical phenotyping, endocrine assessment, karyotype analysis, and molecular detection of Y-derived sequences such as SRY by fluorescence quantitative polymerase chain reaction (QF-PCR) and fluorescence in situ hybridization (FISH). However, even if the above technologies are combined, there are still many limitations. These techniques have a limited range of applications and can only detect the presence of SRY genes on the Y chromosome and roughly determine the location of the Y chromosome. In addition, cross-use is not only time-consuming, but also requires multiple tests. CNV-seq and OGM have been used to detect Y chromosome variation and complex Y chromosome rearrangement.\u003c/p\u003e\u003cp\u003eCNV-seq detects the location and size of copy number variation by extracting sample DNA and cutting it into short fragments, then performing high-throughput sequencing, generating a large number of read data, analyzing these data using bioinformatics algorithms, and calculating read count differences within sliding windows\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. CNV-seq can detect copy number variation as low as 100 kb, but cannot identify balanced SVs and insertions and deletions\u0026thinsp;\u0026lt;\u0026thinsp;50 kb\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. By directly imaging fluorescently labeled ultra-high molecular weight (UHMW) DNA, OGM can accurately and automatically identify clinically significant balanced and unbalanced SV in clinical samples. At present, many scientific experiments and clinical confirmatory studies have proved that, as a new generation of cell genomics technology, OGM can detect all kinds of balanced and unbalanced SV and numerical abnormalities as low as 0.5kb at one time, with high resolution\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,26\u003c/sup\u003e and 100% coincidence rate with clinical standard of care (SOC) in sensitivity, specificity, positive predictive value and negative predictive value\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. However, the same OGM has insufficient detection performance for heterochromatin region\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The combination of the two techniques in this study improved the sensitivity and accuracy of the test results. CNV-seq technology is based on second-generation sequencing and has low cost, but its resolution ability for complex regions (such as repetitive sequences or GC-rich regions) is limited\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, and its detection ability for PAR regions is insufficient\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. OGM technology can accurately resolve structural variations and repetitive sequences in PAR regions. The reliability of the results was cross-verified by the two technologies.\u003c/p\u003e\u003cp\u003eAgnethe et al.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003edetected the Y chromosome of 11 patients with 46,XX DSD by long-read sequencing technique, among which 1 patient did not detect SRY gene and other genetic causes leading to male sexual development, and the other 10 patients detected the breakpoints of Y chromosome and X chromosome, and the smallest fragment size was 2,782 kb. Therefore, based on the characteristics of OGM technology, the diagnostic ability of 46,XX DSD patients is similar to that of long-read sequencing technology, and it is also impossible to find SRY-independent pathogenic gene variants such as \u003cem\u003eSOX9\u003c/em\u003e and \u003cem\u003eRSPO1\u003c/em\u003e\u003csup\u003e32\u0026ndash;34\u003c/sup\u003e. Existing studies have shown that CNV-seq and OGM technologies have their own advantages in 46,XX DSD diagnosis. The former can detect large-segment copy number variations while the latter is good at analyzing fine structural variations. However, both of them have limitations in detecting heterochromatin regions and cannot identify non-SRY dependent pathogenic mutations. In general, the combination of OGM and CNV-seq can detect structural variation patterns with high resolution, thus making more accurate and comprehensive cytogenetic diagnosis, which has clinical significance in genetic counseling.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e46,XX testicular DSD is a rare disorder with discrepancies between genetic, gonadal, and phenotypic sex. In this case, CNV-seq identified critical deletions in X and Y chromosomes, while OGM further resolved the complex Y-chromosome rearrangements, including translocations, deletions, and inversions, and precisely localized Y-chromosome ectopy to the X short arm. This study highlights that the combination of OGM and CNV-seq overcomes the limitations of traditional diagnostics, enabling high-resolution characterization of structural variations in 46,XX testicular DSD. Such an integrated approach enhances the accuracy of genetic diagnosis, clarifies the pathogenic mechanism, and provides valuable insights for clinical management and genetic counseling in sex reversal syndrome.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthical Approval\u003c/h2\u003e\u003cp\u003eApproval for this study was obtained from the Ethics Review Committee of Wenzhou People\u0026rsquo;s Hospital (Approval No.KY-202508-035).\u003c/p\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eNo competing interest between the authors.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis study was funded by Wenzhou City Major Scientific and Technological innovation Project (ZY2024023), and Basic Research Project of Wenzhou City (Y2023528).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eHW and ZX drafted the manuscript. XZ and HP conducted the experiments and data analysis. JZ designed and supervised the study. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eWe would like to thank the patient and his family for their participation and cooperation in this study. We also acknowledge the support of the staff at the Center for Reproductive Medicine, Wenzhou People\u0026rsquo;s Hospital.\u003c/p\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and/or analyzed during the current study are not publicly available due to patient confidentiality and privacy regulations but are available from the corresponding author on reasonable request.\u003c/p\u003e\u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlves C, Braid Z, Coeli FB, Mello MP. 46,XX male - testicular disorder of sexual differentiation (DSD): hormonal, molecular and cytogenetic studies. 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Identification of the first promoter-specific gain-of-function SOX9 missense variant (p.E50K) in a patient with 46,XX ovotesticular disorder of sex development. American J Med Genet Part A Apr. 2021;185(4):1067\u0026ndash;75. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/ajmg.a.62063\u003c/span\u003e\u003cspan address=\"10.1002/ajmg.a.62063\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Sex Reversal Syndrome, Structural Variation, OGM, CNV-seq, Y-chromosome Translocation","lastPublishedDoi":"10.21203/rs.3.rs-7475133/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7475133/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eTo clarify the chromosomal structural variations in a patient with 46,XX testicular disorder of sex development (DSD) using combined genetic technologies.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eChromosome karyotype analysis and whole exome sequencing (WES) were initially performed in a phenotypically male patient. Further analyses using optical genome mapping (OGM) and CNV-seq were conducted on fresh peripheral blood to characterize structural variations.\u003c/p\u003e\u003ch2\u003eResult\u003c/h2\u003e\u003cp\u003eKaryotyping revealed 46,XX,inv(9)(p12q13), and WES suggested possible Y-chromosome sequences. CNV-seq indicated deletions in Xp22.33, Yp11.31-p11.2, and Yp11.2, confirming 46,XX testicular DSD. OGM futher demonstrated a structural translocation Yp segments to the X chromosome short arm, with three breakage-reconnection events in Yp11.2, including deletions and inversions, clarifying the complex Y-chromosome rearrangements and derivative X-chromosome structure.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eCombined OGM and CNV-seq precisely localized Y-chromosome ectopy to the X short arm and characterized its complex rearrangements, providing patients with more accurate and comprehensive genetic diagnosis, and which has important clinical significance for genetic counseling of sex reversal syndrome.\u003c/p\u003e","manuscriptTitle":"Combined Optical Genome Mapping and CNV-Seq indentify Complex Y-Chromosome Rearrangements and Ectopy in 46,XX Testicular Disorder of Sex Development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-10 16:51:03","doi":"10.21203/rs.3.rs-7475133/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b7ca4c59-d45d-49bc-8466-2bf7a2723195","owner":[],"postedDate":"September 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-13T17:38:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-10 16:51:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7475133","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7475133","identity":"rs-7475133","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}