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Accurate detection of expanded\n                      <jats:italic>FMR1<\/jats:italic>\n                      alleles is essential for timely diagnosis and management. Triplet-repeat primed PCR is the most widely used method for detecting FXS; however, it has limitations in detecting low DNA input (&lt;\u200910\u00a0ng\/\u03bcL) and low-level mosaicism (&lt;\u20095%). This study aimed to develop an improved method for detecting\n                      <jats:italic>FMR1<\/jats:italic>\n                      CGG repeat expansions, outperforming existing methods in efficiency, reliability and sensitivity.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      We developed a novel four-primer PCR with capillary electrophoresis assay (FP-PCR\/CE) and validated its performance in identifying and sizing\n                      <jats:italic>FMR1<\/jats:italic>\n                      alleles using DNA standards and multi-center clinical samples (\n                      <jats:italic>N<\/jats:italic>\n                      \u2009=\u20091690). Comparative analyses were performed against the AmplideX\n                      <jats:italic>FMR1<\/jats:italic>\n                      PCR\/CE assay and Southern blot to assess the accuracy, sensitivity, and clinical reliability of this assay.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The FP-PCR\/CE assay demonstrated 100% concordance with DNA standards for CGG repeat sizing and mosaicism detection. It detected DNA input\u2009\u2265\u20092.5\u00a0ng\/\u03bcL and mosaic alleles at a mass fraction as low as 1%. In clinical validation, FP-PCR\/CE achieved 100% concordance in\n                      <jats:italic>FMR1<\/jats:italic>\n                      allele characterization with both the AmplideX assay and Southern blot, while exhibiting higher sensitivity for detecting mosaicism. Additionally, the assay identified AGG interruptions within\n                      <jats:italic>FMR1<\/jats:italic>\n                      alleles. The FP-PCR\/CE assay also reduced testing time to under 7\u00a0h and lowered the cost to\u2009&lt;\u2009$80 per test.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>\n                      The FP-PCR\/CE assay is a rapid, accurate, and cost-effective method for\n                      <jats:italic>FMR1<\/jats:italic>\n                      CGG repeat analysis, offering improved sensitivity for mosaicism detection. Its scalability and reliability support its potential for broader use in FXS carrier screening, clinical diagnosis and research.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Graphical abstract<\/jats:title>\n                  <\/jats:sec>","DOI":"10.1007\/s12519-025-00977-5","type":"journal-article","created":{"date-parts":[[2025,9,12]],"date-time":"2025-09-12T15:59:51Z","timestamp":1757692791000},"page":"1040-1052","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Enhanced accuracy and sensitivity in detecting FMR1 CGG repeats: a multicenter evaluation of a novel PCR-capillary electrophoresis assay"],"prefix":"10.1007","volume":"21","author":[{"given":"Xin-Yi","family":"Shou","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhi-Wei","family":"Zhu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hua","family":"Jin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ji-Hong","family":"Hu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ti-Zhen","family":"Yan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qing-Yan","family":"Zhong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wen-Hao","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jian-Hua","family":"Mao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Min-Yue","family":"Dong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qiong","family":"Xu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shao-Qing","family":"Ni","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,9,12]]},"reference":[{"key":"977_CR1","doi-asserted-by":"publisher","first-page":"S194","DOI":"10.1542\/peds.2016-1159F","volume":"139","author":"WE Kaufmann","year":"2017","unstructured":"Kaufmann WE, Kidd SA, Andrews HF, Budimirovic DB, Esler A, Haas-Givler B, et al. 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China. No other financial or non-financial benefits have been received or will be received from any party related directly or indirectly to the subject of this article.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Conflict of interest"}},{"value":"The study was conducted in accordance with the Declaration of Helsinki and was approved by the respective institutional ethics committees. The approval numbers are as follows: Children\u2019s Hospital, Zhejiang University School of Medicine: 2020-IEC-003, 2022-IEC-0034-P-01; Women\u2019s Hospital, Zhejiang University School of Medicine: IRB-20200079-K; Jinan Maternity and Child Care Hospital: IEC20200301; Liuzhou Maternity and Child Healthcare Hospital: YJ202101401; Children\u2019s Hospital of Fudan University: IRB[2022]389; Hunan Children\u2019s Hospital: HNSETYYLL2023005. This study is registered under the trial number ChiCTR2500102175. All enrolled patients provided written informed consent for molecular analyses prior to the collection of tissue specimens. The use of residual EDTA-anticoagulated peripheral blood from previous clinical tests was exempt from informed consent. Test results were communicated to the patients\u2019 physicians, who incorporated the genetic findings with clinical presentations to establish a final diagnosis.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethical approval"}}]}}