{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,30]],"date-time":"2026-10-30T13:18:28Z","timestamp":1793366308195,"version":"4.5.0"},"reference-count":38,"publisher":"Oxford University Press (OUP)","issue":"12","license":[{"start":{"date-parts":[[2022,9,29]],"date-time":"2022-09-29T00:00:00Z","timestamp":1664409600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2022,12,6]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>Fragile X syndrome (FXS) is the most frequent cause of inherited X-linked intellectual disability. Conventional FXS genetic testing methods mainly focus on FMR1 CGG expansions and fail to identify AGG interruptions, rare intragenic variants, and large gene deletions.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>A long-range PCR and long-read sequencing-based assay termed comprehensive analysis of FXS (CAFXS) was developed and evaluated in Coriell and clinical samples by comparing to Southern blot analysis and triplet repeat\u2013primed PCR (TP\u2013PCR).<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>CAFXS accurately detected the number of CGG repeats in the range of 93 to at least 940 with mass fraction of 0.5% to 1% in the background of normal alleles, which was 2\u20134-fold analytically more sensitive than TP\u2013PCR. All categories of mutations detected by control methods, including full mutations in 30 samples, were identified by CAFXS for all 62 clinical samples. CAFXS accurately determined AGG interruptions in all 133 alleles identified, even in mosaic alleles. CAFXS successfully identified 2 rare intragenic variants including the c.879A &amp;gt; C variant in exon 9 and a 697-bp microdeletion flanking upstream of CGG repeats, which disrupted primer annealing in TP\u2013PCR assay. In addition, CAFXS directly determined the breakpoints of a 237.1-kb deletion and a 774.0-kb deletion encompassing the entire FMR1 gene in 2 samples.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>Long-read sequencing-based CAFXS represents a comprehensive assay for identifying FMR1 CGG expansions, AGG interruptions, rare intragenic variants, and large gene deletions, which greatly improves the genetic screening and diagnosis for FXS.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1093\/clinchem\/hvac154","type":"journal-article","created":{"date-parts":[[2022,8,24]],"date-time":"2022-08-24T09:56:41Z","timestamp":1661335001000},"page":"1529-1540","source":"Crossref","is-referenced-by-count":41,"title":["Comprehensive Analysis of Fragile X Syndrome: Full Characterization of the\n                    <i>FMR1<\/i>\n                    Locus by Long-Read Sequencing"],"prefix":"10.1093","volume":"68","author":[{"given":"Qiaowei","family":"Liang","sequence":"first","affiliation":[{"name":"Department of Medical Genetics, Hunan Jiahui Genetics Hospital , Changsha, Hunan , China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingdi","family":"Liu","sequence":"additional","affiliation":[{"name":"Center for Medical Genetics and Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University , Changsha, Hunan , China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yaning","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Medical Genetics, Hunan Jiahui Genetics Hospital , Changsha, Hunan , China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ranhui","family":"Duan","sequence":"additional","affiliation":[{"name":"Center for Medical Genetics and Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University , Changsha, Hunan , China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wanli","family":"Meng","sequence":"additional","affiliation":[{"name":"Berry Genomics Corporation , Beijing , China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiahan","family":"Zhan","sequence":"additional","affiliation":[{"name":"Berry Genomics Corporation , Beijing , China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiahui","family":"Xia","sequence":"additional","affiliation":[{"name":"Department of Medical Genetics, Hunan Jiahui Genetics Hospital , Changsha, Hunan , China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aiping","family":"Mao","sequence":"additional","affiliation":[{"name":"Berry Genomics Corporation , Beijing , China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Desheng","family":"Liang","sequence":"additional","affiliation":[{"name":"Department of Medical Genetics, Hunan Jiahui Genetics Hospital , Changsha, Hunan , China"},{"name":"Center for Medical Genetics and Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University , Changsha, Hunan , China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lingqian","family":"Wu","sequence":"additional","affiliation":[{"name":"Department of Medical Genetics, Hunan Jiahui Genetics Hospital , Changsha, Hunan , China"},{"name":"Center for Medical Genetics and Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University , Changsha, Hunan , China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2022,9,29]]},"reference":[{"key":"2022120612243912700_hvac154-B1","doi-asserted-by":"crossref","first-page":"784","DOI":"10.1126\/science.256.5058.784","article-title":"Triplet repeat mutations in human disease","volume":"256","author":"Caskey","year":"1992","journal-title":"Science"},{"key":"2022120612243912700_hvac154-B2","doi-asserted-by":"crossref","first-page":"1218","DOI":"10.3390\/genes12081218","article-title":"FMR1 and autism, an intriguing connection revisited","volume":"12","author":"Fyke","year":"2021","journal-title":"Genes"},{"key":"2022120612243912700_hvac154-B3","doi-asserted-by":"crossref","first-page":"799","DOI":"10.1038\/s41436-021-01115-y","article-title":"Committee ALQA","volume":"23","author":"Spector","year":"2021","journal-title":"Genet Med"},{"key":"2022120612243912700_hvac154-B4","doi-asserted-by":"crossref","first-page":"454","DOI":"10.1086\/367713","article-title":"Expansion of the fragile X CGG repeat in females with premutation or intermediate alleles","volume":"72","author":"Nolin","year":"2003","journal-title":"Am J Hum Genet"},{"key":"2022120612243912700_hvac154-B5","doi-asserted-by":"crossref","first-page":"729","DOI":"10.1038\/gim.2012.34","article-title":"AGG interruptions within the maternal FMR1 gene reduce the risk of offspring with fragile X syndrome","volume":"14","author":"Yrigollen","year":"2012","journal-title":"Genet Med"},{"key":"2022120612243912700_hvac154-B6","doi-asserted-by":"crossref","first-page":"358","DOI":"10.1038\/gim.2014.106","article-title":"Fragile X full mutation expansions are inhibited by one or more AGG interruptions in premutation carriers","volume":"17","author":"Nolin","year":"2015","journal-title":"Genet Med"},{"key":"2022120612243912700_hvac154-B7","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1186\/1866-1955-6-24","article-title":"AGG interruptions and maternal age affect FMR1 CGG repeat allele stability during transmission","volume":"6","author":"Yrigollen","year":"2014","journal-title":"J Neurodev Disord"},{"key":"2022120612243912700_hvac154-B8","doi-asserted-by":"crossref","first-page":"1358","DOI":"10.1002\/ajmg.a.32261","article-title":"Mosaic FMR1 deletion causes fragile X syndrome and can lead to molecular misdiagnosis: a case report and review of the literature","volume":"146A","author":"Coffee","year":"2008","journal-title":"Am J Med Genet A"},{"key":"2022120612243912700_hvac154-B9","doi-asserted-by":"crossref","first-page":"2512","DOI":"10.1002\/ajmg.a.33626","article-title":"Identification of novel FMR1 variants by massively parallel sequencing in developmentally delayed males","volume":"152A","author":"Collins","year":"2010","journal-title":"Am J Med Genet A"},{"key":"2022120612243912700_hvac154-B10","doi-asserted-by":"crossref","first-page":"1669","DOI":"10.3390\/genes12111669","article-title":"Beyond trinucleotide repeat expansion in fragile X syndrome: rare coding and noncoding variants in FMR1 and associated phenotypes","volume":"12","author":"Tekendo-Ngongang","year":"2021","journal-title":"Genes"},{"key":"2022120612243912700_hvac154-B11","doi-asserted-by":"crossref","first-page":"423","DOI":"10.1038\/ejhg.2016.204","article-title":"Intragenic FMR1 disease-causing variants: a significant mutational mechanism leading to Fragile-X syndrome","volume":"25","author":"Quartier","year":"2017","journal-title":"Eur J Hum Genet"},{"key":"2022120612243912700_hvac154-B12","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1002\/ajmg.a.38504","article-title":"Rare FMR1 gene mutations causing fragile X syndrome: a review","volume":"176","author":"Sitzmann","year":"2018","journal-title":"Am J Med Genet A"},{"key":"2022120612243912700_hvac154-B13","doi-asserted-by":"crossref","first-page":"743230","DOI":"10.3389\/fgene.2021.743230","article-title":"Characterization of FMR1 repeat expansion and intragenic variants by indirect sequence capture","volume":"12","author":"Grosso","year":"2021","journal-title":"Front Genet"},{"key":"2022120612243912700_hvac154-B14","doi-asserted-by":"crossref","first-page":"7407","DOI":"10.1074\/jbc.R800024200","article-title":"Mutation spectra in fragile X syndrome induced by deletions of CGG*CCG repeats","volume":"284","author":"Wells","year":"2009","journal-title":"J Biol Chem"},{"key":"2022120612243912700_hvac154-B15","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1038\/ejhg.2014.185","article-title":"EMQN best practice guidelines for the molecular genetic testing and reporting of fragile X syndrome and other fragile X-associated disorders","volume":"23","author":"Biancalana","year":"2015","journal-title":"Eur J Hum Genet"},{"key":"2022120612243912700_hvac154-B16","doi-asserted-by":"crossref","first-page":"1047","DOI":"10.1016\/0092-8674(91)90283-5","article-title":"Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox","volume":"67","author":"Fu","year":"1991","journal-title":"Cell"},{"key":"2022120612243912700_hvac154-B17","doi-asserted-by":"crossref","first-page":"589","DOI":"10.2353\/jmoldx.2010.090227","article-title":"An information-rich CGG repeat primed PCR that detects the full range of fragile X expanded alleles and minimizes the need for southern blot analysis","volume":"12","author":"Chen","year":"2010","journal-title":"J Mol Diagn"},{"key":"2022120612243912700_hvac154-B18","doi-asserted-by":"crossref","first-page":"505","DOI":"10.2353\/jmoldx.2010.090229","article-title":"A simple, high-throughput assay for Fragile X expanded alleles using triple repeat primed PCR and capillary electrophoresis","volume":"12","author":"Lyon","year":"2010","journal-title":"J Mol Diagn"},{"key":"2022120612243912700_hvac154-B19","doi-asserted-by":"crossref","first-page":"162","DOI":"10.1097\/GIM.0b013e3181d0d40e","article-title":"Qualitative assessment of FMR1 (CGG)n triplet repeat status in normal, intermediate, premutation, full mutation, and mosaic carriers in both sexes: implications for fragile X syndrome carrier and newborn screening","volume":"12","author":"Hantash","year":"2010","journal-title":"Genet Med"},{"key":"2022120612243912700_hvac154-B20","doi-asserted-by":"crossref","first-page":"399","DOI":"10.1373\/clinchem.2009.136101","article-title":"A novel FMR1 PCR method for the routine detection of low abundance expanded alleles and full mutations in fragile X syndrome","volume":"56","author":"Filipovic-Sadic","year":"2010","journal-title":"Clin Chem"},{"key":"2022120612243912700_hvac154-B21","doi-asserted-by":"crossref","first-page":"2","DOI":"10.2353\/jmoldx.2008.070105","article-title":"Consensus characterization of 16 FMR1 reference materials: a consortium study","volume":"10","author":"Amos Wilson","year":"2008","journal-title":"J Mol Diagn"},{"key":"2022120612243912700_hvac154-B22","doi-asserted-by":"crossref","first-page":"43","DOI":"10.2353\/jmoldx.2008.070073","article-title":"A rapid polymerase chain reaction-based screening method for identification of all expanded alleles of the fragile X (FMR1) gene in newborn and high-risk populations","volume":"10","author":"Tassone","year":"2008","journal-title":"J Mol Diagn"},{"key":"2022120612243912700_hvac154-B23","doi-asserted-by":"crossref","first-page":"476","DOI":"10.1016\/j.jmoldx.2012.03.005","article-title":"Performance evaluation of 2 methods using commercially available reagents for PCR-based detection of FMR1 mutation","volume":"14","author":"Juusola","year":"2012","journal-title":"J Mol Diagn"},{"key":"2022120612243912700_hvac154-B24","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1093\/hmg\/4.1.45","article-title":"Hotspot for deletions in the CGG repeat region of FMR1 in fragile X patients","volume":"4","author":"de Graaff","year":"1995","journal-title":"Hum Mol Genet"},{"key":"2022120612243912700_hvac154-B25","doi-asserted-by":"crossref","first-page":"624","DOI":"10.1136\/jmg.38.9.624","article-title":"Microdeletion in the FMR-1 gene: an apparent null allele using routine clinical PCR amplification","volume":"38","author":"Hegde","year":"2001","journal-title":"J Med Genet"},{"key":"2022120612243912700_hvac154-B26","doi-asserted-by":"crossref","first-page":"828","DOI":"10.1016\/j.jmoldx.2017.06.008","article-title":"Improved assays for AGG interruptions in Fragile X premutation carriers","volume":"19","author":"Hayward","year":"2017","journal-title":"J Mol Diagn"},{"key":"2022120612243912700_hvac154-B27","doi-asserted-by":"crossref","first-page":"736","DOI":"10.12688\/f1000research.13980.1","article-title":"Recent advances in the detection of repeat expansions with short-read next-generation sequencing","volume":"7","author":"Bahlo","year":"2018","journal-title":"F1000Res"},{"key":"2022120612243912700_hvac154-B28","doi-asserted-by":"crossref","first-page":"234","DOI":"10.1016\/S1474-4422(21)00462-2","article-title":"Whole genome sequencing for the diagnosis of neurological repeat expansion disorders in the UK: a retrospective diagnostic accuracy and prospective clinical validation study","volume":"21","author":"Ibanez","year":"2022","journal-title":"Lancet Neurol"},{"key":"2022120612243912700_hvac154-B29","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1101\/gr.141705.112","article-title":"Sequencing the unsequenceable: expanded CGG-repeat alleles of the fragile X gene","volume":"23","author":"Loomis","year":"2013","journal-title":"Genome Res"},{"key":"2022120612243912700_hvac154-B30","doi-asserted-by":"crossref","first-page":"324","DOI":"10.1002\/humu.23150","article-title":"Detecting AGG interruptions in male and female FMR1 premutation carriers by single-molecule sequencing","volume":"38","author":"Ardui","year":"2017","journal-title":"Hum Mutat"},{"key":"2022120612243912700_hvac154-B31","doi-asserted-by":"crossref","first-page":"150","DOI":"10.3389\/fgene.2018.00150","article-title":"Detecting AGG interruptions in females with a FMR1 premutation by long-read single-molecule sequencing: a 1 year clinical experience","volume":"9","author":"Ardui","year":"2018","journal-title":"Front Genet"},{"key":"2022120612243912700_hvac154-B32","doi-asserted-by":"crossref","first-page":"1195","DOI":"10.1016\/j.jmoldx.2021.06.008","article-title":"A more universal approach to comprehensive analysis of thalassemia alleles (CATSA)","volume":"23","author":"Liang","year":"2021","journal-title":"J Mol Diagn"},{"key":"2022120612243912700_hvac154-B33","doi-asserted-by":"crossref","first-page":"803","DOI":"10.1177\/0883073814538508","article-title":"Mutational analyses of the FMR1 gene in Chinese pediatric population of fragile X suspects: low tolerance for point mutation","volume":"30","author":"Luo","year":"2015","journal-title":"J Child Neurol"},{"key":"2022120612243912700_hvac154-B34","doi-asserted-by":"crossref","first-page":"1262","DOI":"10.1002\/humu.23580","article-title":"Detailed analysis of HTT repeat elements in human blood using targeted amplification-free long-read sequencing","volume":"39","author":"Hoijer","year":"2018","journal-title":"Hum Mutat"},{"key":"2022120612243912700_hvac154-B35","doi-asserted-by":"crossref","first-page":"1478","DOI":"10.1038\/s41587-019-0293-x","article-title":"Analysis of short tandem repeat expansions and their methylation state with nanopore sequencing","volume":"37","author":"Giesselmann","year":"2019","journal-title":"Nat Biotechnol"},{"key":"2022120612243912700_hvac154-B36","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1038\/s10038-021-00983-1","article-title":"The value of single-molecule real-time technology in the diagnosis of rare thalassemia variants and analysis of phenotype-genotype correlation","volume":"67","author":"Luo","year":"2022","journal-title":"J Hum Genet"},{"key":"2022120612243912700_hvac154-B37","doi-asserted-by":"crossref","first-page":"927","DOI":"10.1093\/clinchem\/hvac046","article-title":"Comprehensive analysis of congenital adrenal hyperplasia using long-read sequencing","volume":"68","author":"Liu","year":"2022","journal-title":"Clin Chem"},{"key":"2022120612243912700_hvac154-B38","doi-asserted-by":"crossref","first-page":"P1009","DOI":"10.1016\/j.jmoldx.2022.05.001","article-title":"Comprehensive analysis of spinal muscular atrophy: SMN1 copy number, intragenic mutation, and 2 + 0 carrier analysis by third-generation sequencing","volume":"24","author":"Li","year":"2022","journal-title":"J Mol Diagn"}],"container-title":["Clinical Chemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/clinchem\/article-pdf\/68\/12\/1529\/47615854\/hvac154.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/clinchem\/article-pdf\/68\/12\/1529\/47615854\/hvac154.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,6]],"date-time":"2022-12-06T18:35:13Z","timestamp":1670351713000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/clinchem\/article\/68\/12\/1529\/6726663"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,9,29]]},"references-count":38,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2022,9,29]]},"published-print":{"date-parts":[[2022,12,6]]}},"URL":"https:\/\/doi.org\/10.1093\/clinchem\/hvac154","relation":{},"ISSN":["0009-9147","1530-8561"],"issn-type":[{"value":"0009-9147","type":"print"},{"value":"1530-8561","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2022,12,2]]},"published":{"date-parts":[[2022,9,29]]}}}