{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T10:23:06Z","timestamp":1789467786234,"version":"build-2803163510"},"reference-count":31,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2023,9,14]],"date-time":"2023-09-14T00:00:00Z","timestamp":1694649600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Genet."],"abstract":"<jats:p><jats:bold>Background:<\/jats:bold> This study aimed to perform preimplantation genetic testing (PGT) for a female Coffin-Lowry Syndrome (CLS) patient with a <jats:italic>de novo<\/jats:italic> mutation (DNM) in RPS6KA3. It was challenging to establish the haplotype in this family because of the lack of information from affected family members. Hence, we explored a new and reliable strategy for the detection of the DNM in PGT, using Oxford Nanopore Technologies (ONT) and the MARSALA platform.<\/jats:p><jats:p><jats:bold>Methods:<\/jats:bold> We performed whole-exome sequencing (WES) on the proband and confirmed the pathogenic mutation by Sanger sequencing. The proband then underwent PGT to prevent the transmission of the pathogenic mutation to her offspring. We diverged from the conventional methods and used long-read sequencing (LRS) on the ONT platform to directly detect the mutation and nearby SNPs, for construction of the haplotype in the preclinical phase of PGT. In the clinical phase of embryo diagnosis, the MARSALA method was used to detect both the SNP-based haplotype and chromosome copy number variations (CNVs), in each blastocyst. Finally, a normal embryo was selected by comparison to the haplotype of the proband and transferred into the uterus. Sanger sequencing and karyotyping were performed by amniocentesis, at 17\u00a0weeks of gestation, to confirm the accuracy of PGT.<\/jats:p><jats:p><jats:bold>Results:<\/jats:bold> Using WES, we found the novel, heterozygous, pathogenic c.1496delG (p.Gly499Valfs*25) mutation of RPS6KA3 in the proband. The SNP-based haplotype that was linked to the pathogenic mutation site was successfully established in the proband, without the need for other family members to be tested with ONT. Eight blastocysts were biopsied to perform PGT and were assessed with a haplotype linkage analysis (30 SNP sites selected), to give results that were consistent with direct mutation detection using Sanger sequencing. The results of PGT showed that three of the eight blastocysts were normal, without the DNM. Moreover, the patient had a successful pregnancy, after transfer of a normal blastocyst into the uterus, and delivered a healthy baby.<\/jats:p><jats:p><jats:bold>Conclusion:<\/jats:bold> The ONT platform, combined with the MARSALA method, can be used to perform PGT for DNM patients without the need for other samples as a reference.<\/jats:p>","DOI":"10.3389\/fgene.2023.1169868","type":"journal-article","created":{"date-parts":[[2023,9,14]],"date-time":"2023-09-14T20:03:14Z","timestamp":1694721794000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":7,"title":["Establishment of linkage phase, using Oxford Nanopore Technologies, for preimplantation genetic testing of Coffin-Lowry syndrome with a de novo RPS6KA3 mutation"],"prefix":"10.3389","volume":"14","author":[{"given":"Xiaojun","family":"Wen","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing","family":"Du","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhiming","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nengqing","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junye","family":"Huo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jieliang","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wanna","family":"Ke","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiaqi","family":"Wu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaowu","family":"Fang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiufeng","family":"Lin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2023,9,14]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"75","DOI":"10.1080\/19396368.2018.1472315","article-title":"The clinical application of single-sperm-based SNP haplotyping for PGD of osteogenesis imperfecta","volume":"65","author":"Chen","year":"2019","journal-title":"Syst. Biol. Reprod. Med."},{"key":"B2","doi-asserted-by":"publisher","first-page":"774","DOI":"10.1016\/j.fertnstert.2021.04.012","article-title":"Variant haplophasing by long-read sequencing: a new approach to preimplantation genetic testing workups","volume":"116","author":"Cheng","year":"2021","journal-title":"Fertil. Steril."},{"key":"B3","doi-asserted-by":"publisher","first-page":"900226","DOI":"10.3389\/fgene.2022.900226","article-title":"Case report: chinese female patients with a heterozygous pathogenic RPS6KA3 gene variant c.898C>T and distal 22q11.2 microdeletion","volume":"13","author":"Cong","year":"2022","journal-title":"Front. Genet."},{"key":"B4","doi-asserted-by":"publisher","first-page":"161","DOI":"10.1111\/j.1399-0004.2006.00660.x","article-title":"Identification of novel mutations in the RSK2 gene (RPS6KA3) in patients with Coffin-Lowry syndrome","volume":"70","author":"Delaunoy","year":"2006","journal-title":"Clin. Genet."},{"key":"B5","doi-asserted-by":"publisher","first-page":"298","DOI":"10.1080\/19396368.2021.1926574","article-title":"Affected-embryo-based SNP haplotyping with NGS for the preimplantation genetic testing of Marfan syndrome","volume":"67","author":"Deng","year":"2021","journal-title":"Syst. Biol. Reprod. Med."},{"key":"B6","doi-asserted-by":"publisher","first-page":"307","DOI":"10.1097\/00001703-199906000-00013","article-title":"Culture and transfer of human blastocysts","volume":"11","author":"Gardner","year":"1999","journal-title":"Curr. Opin. Obstet. Gynecol."},{"key":"B7","doi-asserted-by":"publisher","first-page":"651","DOI":"10.1136\/jmg.2009.069971","article-title":"Karyomapping: a universal method for genome wide analysis of genetic disease based on mapping crossovers between parental haplotypes","volume":"47","author":"Handyside","year":"2010","journal-title":"J. Med. Genet."},{"key":"B8","doi-asserted-by":"publisher","first-page":"33","DOI":"10.1093\/humrep\/deq231","article-title":"ESHRE PGD consortium best practice guidelines for amplification-based PGD","volume":"26","author":"Harton","year":"2011","journal-title":"Hum. Reprod. Oxf. Engl."},{"key":"B9","doi-asserted-by":"publisher","first-page":"63","DOI":"10.1016\/j.rbmo.2021.09.008","article-title":"The clinical application of single-sperm-based single-nucleotide polymorphism haplotyping for PGT of patients with genetic diseases","volume":"44","author":"Huang","year":"2022","journal-title":"Reprod. Biomed. Online"},{"key":"B10","doi-asserted-by":"publisher","first-page":"519","DOI":"10.1038\/nature24018","article-title":"Parental influence on human germline de novo mutations in 1,548 trios from Iceland","volume":"549","author":"J\u00f3nsson","year":"2017","journal-title":"Nature"},{"key":"B11","doi-asserted-by":"publisher","first-page":"117","DOI":"10.1186\/s12920-020-00766-1","article-title":"Haplotyping by linked-read sequencing (HLRS) of the genetic disease carriers for preimplantation genetic testing without a proband or relatives","volume":"13","author":"Li","year":"2020","journal-title":"BMC Med. Genomics"},{"key":"B12","doi-asserted-by":"publisher","first-page":"357","DOI":"10.1007\/s10815-020-02009-9","article-title":"Third-generation sequencing: any future opportunities for PGT?","volume":"38","author":"Liu","year":"2021","journal-title":"J. Assist. Reprod. Genet."},{"key":"B13","doi-asserted-by":"publisher","first-page":"e22267","DOI":"10.1002\/jcla.22267","article-title":"The performance of MALBAC and MDA methods in the identification of concurrent mutations and aneuploidy screening to diagnose beta-thalassaemia disorders at the single- and multiple-cell levels","volume":"32","author":"Liu","year":"2018","journal-title":"J. Clin. Lab. Anal."},{"key":"B14","doi-asserted-by":"publisher","first-page":"668","DOI":"10.1016\/j.fertnstert.2021.06.055","article-title":"Variant haplophasing by long-read sequencing: proof of concept in preimplantation genetic workup and an opportunity to distinguish balanced and normal embryos","volume":"116","author":"Margolis","year":"2021","journal-title":"Fertil. Steril."},{"key":"B15","doi-asserted-by":"publisher","first-page":"75","DOI":"10.1186\/s12958-022-00948-9","article-title":"Using affected embryos to establish linkage phase in preimplantation genetic testing for thalassemia","volume":"20","author":"Ou","year":"2022","journal-title":"Reprod. Biol. Endocrinol."},{"key":"B16","doi-asserted-by":"publisher","first-page":"810900","DOI":"10.3389\/fgene.2021.810900","article-title":"Identifying balanced chromosomal translocations in human embryos by Oxford Nanopore Sequencing and Breakpoints Region analysis","volume":"12","author":"Pei","year":"2021","journal-title":"Front. Genet."},{"key":"B17","doi-asserted-by":"publisher","first-page":"627","DOI":"10.1038\/ejhg.2009.189","article-title":"Coffin-Lowry syndrome","volume":"18","author":"Pereira","year":"2010","journal-title":"Eur. J. Hum. Genet."},{"key":"B18","doi-asserted-by":"publisher","first-page":"72","DOI":"10.1016\/j.rbmo.2010.09.016","article-title":"First successful application of preimplantation genetic diagnosis and haplotyping for congenital hyperinsulinism","volume":"22","author":"Qubbaj","year":"2011","journal-title":"Reprod. Biomed. Online"},{"key":"B19","doi-asserted-by":"publisher","first-page":"405","DOI":"10.1038\/gim.2015.30","article-title":"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","volume":"17","author":"Richards","year":"2015","journal-title":"Genet. Med."},{"key":"B20","doi-asserted-by":"publisher","first-page":"67","DOI":"10.6065\/apem.2142134.067","article-title":"First female Korean child with coffin-lowry syndrome: A novel variant in RPS6KA3 diagnosed by exome sequencing and a literature review","volume":"2022","author":"Song","year":"2022","journal-title":"Ann. Pediatr. Endocrinol. Metab."},{"key":"B21","doi-asserted-by":"publisher","first-page":"986","DOI":"10.1093\/humrep\/deaa034","article-title":"The status of preimplantation genetic testing in the UK and USA","volume":"35","author":"Theobald","year":"2020","journal-title":"Hum. Reprod. Oxf. Engl."},{"key":"B22","doi-asserted-by":"publisher","first-page":"3136","DOI":"10.12659\/msm.904546","article-title":"De novo paternal FBN1 mutation detected in embryos before implantation","volume":"23","author":"Wang","year":"2017","journal-title":"Med. Sci. Monit."},{"key":"B23","doi-asserted-by":"publisher","first-page":"2425","DOI":"10.1007\/s10815-021-02188-z","article-title":"A comprehensive PGT-M strategy for ADPKD patients with de novo PKD1 mutations using affected embryo or gametes as proband","volume":"38","author":"Wang","year":"2021","journal-title":"J. Assist. Reprod. Genet."},{"key":"B24","doi-asserted-by":"publisher","first-page":"1155","DOI":"10.1038\/s41587-019-0217-9","article-title":"Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome","volume":"37","author":"Wenger","year":"2019","journal-title":"Nat. Biotechnol."},{"key":"B25","doi-asserted-by":"publisher","first-page":"1146","DOI":"10.1002\/pd.5156","article-title":"A novel approach using long-read sequencing and ddPCR to investigate gonadal mosaicism and estimate recurrence risk in two families with developmental disorders","volume":"37","author":"Wilbe","year":"2017","journal-title":"Prenat. Diagn"},{"key":"B26","doi-asserted-by":"publisher","first-page":"1221","DOI":"10.1093\/humrep\/den488","article-title":"The causes of misdiagnosis and adverse outcomes in PGD","volume":"24","author":"Wilton","year":"2009","journal-title":"Hum. Reprod. Oxf. Engl."},{"key":"B27","doi-asserted-by":"publisher","first-page":"739","DOI":"10.1007\/s10815-022-02415-1","article-title":"Long-read sequencing on the SMRT platform enables efficient haplotype linkage analysis in preimplantation genetic testing for \u03b2-thalassemia","volume":"39","author":"Wu","year":"2022","journal-title":"J. Assist. Reprod. Genet."},{"key":"B28","doi-asserted-by":"publisher","first-page":"1071","DOI":"10.1007\/s10815-018-1158-9","article-title":"Genotyping single-sperm cells by universal MARSALA enables the acquisition of linkage information for combined pre-implantation genetic diagnosis and genome screening","volume":"35","author":"Wu","year":"2018","journal-title":"J. Assist. Reprod. Genet."},{"key":"B29","doi-asserted-by":"publisher","first-page":"1263","DOI":"10.1007\/s10815-019-01451-8","article-title":"Bayesian model for accurate MARSALA (mutated allele revealed by sequencing with aneuploidy and linkage analyses)","volume":"36","author":"Xiong","year":"2019","journal-title":"J. Assist. Reprod. Genet."},{"key":"B30","doi-asserted-by":"publisher","first-page":"15964","DOI":"10.1073\/pnas.1523297113","article-title":"Live births after simultaneous avoidance of monogenic diseases and chromosome abnormality by next-generation sequencing with linkage analyses","volume":"112","author":"Yan","year":"2015","journal-title":"Proc. Natl. Proc. Natl. Acad. Sci. U. S. A."},{"key":"B31","doi-asserted-by":"publisher","first-page":"2525","DOI":"10.1007\/s10815-020-01921-4","article-title":"A whole-genome sequencing-based novel preimplantation genetic testing method for de novo mutations combined with chromosomal balanced translocations","volume":"37","author":"Yuan","year":"2020","journal-title":"J. Assist. Reprod. Genet."}],"container-title":["Frontiers in Genetics"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fgene.2023.1169868\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,9,14]],"date-time":"2023-09-14T20:03:17Z","timestamp":1694721797000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fgene.2023.1169868\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,9,14]]},"references-count":31,"alternative-id":["10.3389\/fgene.2023.1169868"],"URL":"https:\/\/doi.org\/10.3389\/fgene.2023.1169868","relation":{},"ISSN":["1664-8021"],"issn-type":[{"value":"1664-8021","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,9,14]]},"article-number":"1169868"}}