{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T02:10:46Z","timestamp":1760235046470,"version":"build-2065373602"},"reference-count":35,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2021,7,23]],"date-time":"2021-07-23T00:00:00Z","timestamp":1626998400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["JP19K16528"],"award-info":[{"award-number":["JP19K16528"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Tokumori Yasumoto Memorial Trust for Researches on TSC and Related Rare Neurological Dis-eases","award":["2019"],"award-info":[{"award-number":["2019"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["CIMB"],"abstract":"<jats:p>Elaborate analyses of the status of gene mutations in neurofibromatosis type 1 (NF1) are still difficult nowadays due to the large gene sizes, broad mutation spectrum, and the various effects of mutations on mRNA splicing. These problems cannot be solved simply by sequencing the entire coding region using next-generation sequencing (NGS). We recently developed a new strategy, named combined long amplicon sequencing (CoLAS), which is a method for simultaneously analysing the whole genomic DNA region and, also, the full-length cDNA of the disease-causative gene with long-range PCR-based NGS. In this study, CoLAS was specifically arranged for NF1 genetic analysis, then applied to 20 patients (five previously reported and 15 newly recruited patients, including suspicious cases) for optimising the method and to verify its efficacy and benefits. Among new cases, CoLAS detected not only 10 mutations, including three unreported mutations and one mosaic mutation, but also various splicing abnormalities and allelic expression ratios quantitatively. In addition, heterozygous mapping by polymorphisms, including introns, showed copy number monitoring of the entire NF1 gene region was possible in the majority of patients tested. Moreover, it was shown that, when a chromosomal level microdeletion was suspected from heterozygous mapping, it could be detected directly by breakpoint-specific long PCR. In conclusion, CoLAS not simply detect the causative mutation but accurately elucidated the entire structure of the NF1 gene, its mRNA expression, and also the splicing status, which reinforces its high usefulness in the gene analysis of NF1.<\/jats:p>","DOI":"10.3390\/cimb43020057","type":"journal-article","created":{"date-parts":[[2021,7,23]],"date-time":"2021-07-23T10:31:44Z","timestamp":1627036304000},"page":"782-801","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Application of Combined Long Amplicon Sequencing (CoLAS) for Genetic Analysis of Neurofibromatosis Type 1: A Pilot Study"],"prefix":"10.3390","volume":"43","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3189-8111","authenticated-orcid":false,"given":"Sumihito","family":"Togi","sequence":"first","affiliation":[{"name":"Center for Clinical Genomics, Kanazawa Medical University Hospital, Uchinada, Ishikawa 920-0923, Japan"},{"name":"Division of Genomic Medicine, Department of Advanced Medicine, Medical Research Institute, Kanazawa Medical University, Uchinada, Ishikawa 920-0923, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7600-3481","authenticated-orcid":false,"given":"Hiroki","family":"Ura","sequence":"additional","affiliation":[{"name":"Center for Clinical Genomics, Kanazawa Medical University Hospital, Uchinada, Ishikawa 920-0923, Japan"},{"name":"Division of Genomic Medicine, Department of Advanced Medicine, Medical Research Institute, Kanazawa Medical University, Uchinada, Ishikawa 920-0923, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5641-7962","authenticated-orcid":false,"given":"Yo","family":"Niida","sequence":"additional","affiliation":[{"name":"Center for Clinical Genomics, Kanazawa Medical University Hospital, Uchinada, Ishikawa 920-0923, Japan"},{"name":"Division of Genomic Medicine, Department of Advanced Medicine, Medical Research Institute, Kanazawa Medical University, Uchinada, Ishikawa 920-0923, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,7,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1001\/jama.1997.03550010065042","article-title":"The diagnostic evaluation and multidisciplinary management of neurofibromatosis 1 and neurofibromatosis 2","volume":"278","author":"Gutmann","year":"1997","journal-title":"JAMA"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1016\/j.spen.2006.01.007","article-title":"The genetic and molecular pathogenesis of NF1 and NF2","volume":"13","author":"Yohay","year":"2006","journal-title":"Semin. 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