{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,20]],"date-time":"2026-01-20T22:34:48Z","timestamp":1768948488552,"version":"3.49.0"},"reference-count":36,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,1,18]],"date-time":"2026-01-18T00:00:00Z","timestamp":1768694400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National High Level Hospital Clinical Research Funding","award":["2022CR62"],"award-info":[{"award-number":["2022CR62"]}]},{"name":"Peking University Medicine Seed Fund for Interdisciplinary Research","award":["BMU2021MX016"],"award-info":[{"award-number":["BMU2021MX016"]}]},{"name":"Research Fund for the Diagnosis, Treatment, and Management of Rare Diseases","award":["2024-173"],"award-info":[{"award-number":["2024-173"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Fabry disease (FD) exhibits a spectrum of clinical manifestations ranging from mild to severe, posing a diagnostic challenge, particularly in non-classic subtypes. Genetic testing remains a gold standard for a precise diagnosis of FD and is pivotal in genetic counseling. Although conventional approaches such as Sanger sequencing and short-read next-generation sequencing (NGS) have been successfully used to diagnose FD, they often fail to detect deep intronic variants, complex rearrangements, or large deletions or duplications. In contrast, long-read sequencing (LRS) enables comprehensive coverage of intronic and repetitive regions, facilitating precise identification of atypical variants missed by conventional methods. This case series reports two unrelated male patients with clinical, enzymatic, and pathological features consistent with FD, who tested negative for pathogenic variants in the alpha-galactosidase A (GLA) via Sanger sequencing and NGS. LRS identified novel non-coding variants in both patients. Patient 1 carried a ~1.7 kb insertion within intron 4, corresponding to part of a long interspersed nuclear element-1, while RNA sequencing revealed two new GLA transcripts. Patient 2 harbored a ~2.5 kb insertion within a SINE-VNTR-Alu retroposon element located in the 5\u2032-untranslated region, with quantitative real-time PCR showing significantly reduced expression of normal GLA transcripts. These findings reveal non-coding variants that contribute to the missing heritability in FD, highlight this genomic region as a priority for future investigation, and demonstrate the potential utility of LRS in diagnostic workflows for unresolved FD cases.<\/jats:p>","DOI":"10.3390\/ijms27020945","type":"journal-article","created":{"date-parts":[[2026,1,20]],"date-time":"2026-01-20T09:24:34Z","timestamp":1768901074000},"page":"945","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A New Class of Pathogenic Non-Coding Variants in GLA"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-4476-3204","authenticated-orcid":false,"given":"Yujing","family":"Yuan","sequence":"first","affiliation":[{"name":"Department of Neurology, Peking University First Hospital, Beijing 100034, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xinyu","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Neurology, Peking University First Hospital, Beijing 100034, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chen","family":"Ling","sequence":"additional","affiliation":[{"name":"Department of Neurology, Peking University First Hospital, Beijing 100034, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yawen","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Neurology, Peking University First Hospital, Beijing 100034, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Meng","family":"Yu","sequence":"additional","affiliation":[{"name":"Department of Neurology, Peking University First Hospital, Beijing 100034, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhaoxia","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Neurology, Peking University First Hospital, Beijing 100034, China"},{"name":"Beijing Key Laboratory of Neurovascular Diseases, Beijing 100034, China"},{"name":"Rare Disease Medical Center, Peking University First Hospital, Beijing 100034, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yun","family":"Yuan","sequence":"additional","affiliation":[{"name":"Department of Neurology, Peking University First Hospital, Beijing 100034, China"},{"name":"Beijing Key Laboratory of Neurovascular Diseases, Beijing 100034, China"},{"name":"Rare Disease Medical Center, Peking University First Hospital, Beijing 100034, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhiying","family":"Xie","sequence":"additional","affiliation":[{"name":"Department of Neurology, Peking University First Hospital, Beijing 100034, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6241-4207","authenticated-orcid":false,"given":"Wei","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Neurology, Peking University First Hospital, Beijing 100034, China"},{"name":"Beijing Key Laboratory of Neurovascular Diseases, Beijing 100034, China"},{"name":"Rare Disease Medical Center, Peking University First Hospital, Beijing 100034, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"416","DOI":"10.1016\/j.ymgme.2018.02.014","article-title":"Fabry disease revisited: Management and treatment recommendations for adult patients","volume":"123","author":"Ortiz","year":"2018","journal-title":"Mol. Genet. Metab."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"922","DOI":"10.1016\/j.jacc.2020.12.024","article-title":"Cardiac Involvement in Fabry Disease: JACC Review Topic of the Week","volume":"77","author":"Pieroni","year":"2021","journal-title":"J. Am. Coll. Cardiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"641","DOI":"10.1016\/j.amjms.2020.07.011","article-title":"When and How to Diagnose Fabry Disease in Clinical Pratice","volume":"360","author":"Michaud","year":"2020","journal-title":"Am. J. Med. Sci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1600","DOI":"10.1016\/j.ajhg.2023.07.013","article-title":"GLA-modified RNA treatment lowers GB3 levels in iPSC-derived cardiomyocytes from Fabry-affected individuals","volume":"110","author":"Parker","year":"2023","journal-title":"Am. J. Hum. Genet."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Tuttolomondo, A., Chimenti, C., Cianci, V., Gallieni, M., Lanzillo, C., La Russa, A., Limongelli, G., Mignani, R., Olivotto, I., and Pieruzzi, F. (2025). Females with Fabry disease: An expert opinion on diagnosis, clinical management, current challenges and unmet needs. Front. Cardiovasc. Med., 12.","DOI":"10.3389\/fcvm.2025.1536114"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1311","DOI":"10.1038\/ejhg.2008.109","article-title":"Unbalanced GLA mRNAs ratio quantified by real-time PCR in Fabry patients\u2019 fibroblasts results in Fabry disease","volume":"16","author":"Filoni","year":"2008","journal-title":"Eur. J. Hum. Genet."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"108","DOI":"10.1002\/humu.1380020208","article-title":"Fabry disease: Detection of gene rearrangements in the human alpha-galactosidase A gene by multiplex PCR amplification","volume":"2","author":"Kornreich","year":"1993","journal-title":"Hum. Mutat."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e000394","DOI":"10.1161\/JAHA.113.000394","article-title":"Risk of death in heart disease is associated with elevated urinary globotriaosylceramide","volume":"3","author":"Schiffmann","year":"2014","journal-title":"J. Am. Heart Assoc."},{"key":"ref_9","first-page":"754","article-title":"Low frequency of Fabry disease in patients with common heart disease","volume":"20","author":"Schiffmann","year":"2018","journal-title":"Genet. Med. Off. J. Am. Coll. Med. Genet."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1016\/j.mcp.2019.03.008","article-title":"Fabry disease: Detection of Alu-mediated exon duplication by NGS","volume":"45","author":"Farr","year":"2019","journal-title":"Mol. Cell. Probes"},{"key":"ref_11","first-page":"5208312","article-title":"Novel GLA Deletion in a Cypriot Female Presenting with Cornea Verticillata","volume":"2016","author":"Georgiou","year":"2016","journal-title":"Case Rep. Genet."},{"key":"ref_12","first-page":"73","article-title":"Fabry disease in a Japanese population-molecular and biochemical characteristics","volume":"17","author":"Sakuraba","year":"2018","journal-title":"Mol. Genet. Metab. Rep."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1016\/j.ymgme.2021.07.006","article-title":"Gene therapy for Fabry disease: Progress, challenges, and outlooks on gene-editing","volume":"134","author":"Domm","year":"2021","journal-title":"Mol. Genet. Metab."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"542","DOI":"10.1136\/jmedgenet-2019-106467","article-title":"Use of a rare disease registry for establishing phenotypic classification of previously unassigned GLA variants: A consensus classification system by a multispecialty Fabry disease genotype-phenotype workgroup","volume":"57","author":"Germain","year":"2020","journal-title":"J. Med. Genet."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1186\/s40246-024-00697-3","article-title":"Long-read sequencing enables comprehensive molecular genetic diagnosis of Fabry disease","volume":"18","author":"Yao","year":"2024","journal-title":"Hum. Genom."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"994","DOI":"10.1086\/339431","article-title":"Alternative splicing in the alpha-galactosidase A gene: Increased exon inclusion results in the Fabry cardiac phenotype","volume":"70","author":"Ishii","year":"2002","journal-title":"Am. J. Hum. Genet."},{"key":"ref_17","unstructured":"(2021). Chinese Fabry Disease Expert Panel. Expert consensus for diagnosis and treatment of Fabry disease in China (2021). Chin. J. Intern. Med., 60, 321\u2013330."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"564","DOI":"10.1002\/humu.22981","article-title":"HGVS Recommendations for the Description of Sequence Variants: 2016 Update","volume":"37","author":"Dalgleish","year":"2016","journal-title":"Hum. Mutat."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"22372","DOI":"10.1038\/s41598-021-01749-7","article-title":"Detection of single nucleotide and copy number variants in the Fabry disease-associated GLA gene using nanopore sequencing","volume":"11","author":"Nowak","year":"2021","journal-title":"Sci. Rep."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1093","DOI":"10.1007\/s00439-017-1809-4","article-title":"Deep intronic mutations and human disease","volume":"136","year":"2017","journal-title":"Hum. Genet."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"6816","DOI":"10.1128\/MCB.20.18.6816-6825.2000","article-title":"Human genomic sequences that inhibit splicing","volume":"20","author":"Fairbrother","year":"2000","journal-title":"Mol. Cell. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1007","DOI":"10.1126\/science.1073774","article-title":"Predictive identification of exonic splicing enhancers in human genes","volume":"297","author":"Fairbrother","year":"2002","journal-title":"Science"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"831","DOI":"10.1016\/j.cell.2004.11.010","article-title":"Systematic identification and analysis of exonic splicing silencers","volume":"119","author":"Wang","year":"2004","journal-title":"Cell"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"5493","DOI":"10.1093\/nar\/gkac270","article-title":"Exonic splicing code and protein binding sites for calcium","volume":"50","author":"Pengelly","year":"2022","journal-title":"Nucleic Acids Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"258","DOI":"10.1016\/j.ymgme.2016.08.007","article-title":"The prevalent deep intronic c. 639+919 G>A GLA mutation causes pseudoexon activation and Fabry disease by abolishing the binding of hnRNPA1 and hnRNP A2\/B1 to a splicing silencer","volume":"119","author":"Palhais","year":"2016","journal-title":"Mol. Genet. Metab."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Xie, Z., Liu, C., Lu, Y., Sun, C., Liu, Y., Yu, M., Shu, J., Meng, L., Deng, J., and Zhang, W. (2022). Exonization of a deep intronic long interspersed nuclear element in Becker muscular dystrophy. Front. Genet., 13.","DOI":"10.3389\/fgene.2022.979732"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Wimmer, K., Callens, T., Wernstedt, A., and Messiaen, L. (2011). The NF1 gene contains hotspots for L1 endonuclease-dependent de novo insertion. PLoS Genet., 7.","DOI":"10.1371\/journal.pgen.1002371"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Alesi, V., Genovese, S., Lepri, F.R., Catino, G., Loddo, S., Orlando, V., Di Tommaso, S., Morgia, A., Martucci, L., and Di Donato, M. (2023). Deep Intronic LINE-1 Insertions in NF1: Expanding the Spectrum of Neurofibromatosis Type 1-Associated Rearrangements. Biomolecules, 13.","DOI":"10.3390\/biom13050725"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1016\/j.trecan.2019.02.011","article-title":"The Untranslated Regions of mRNAs in Cancer","volume":"5","author":"Schuster","year":"2019","journal-title":"Trends Cancer"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1413","DOI":"10.1126\/science.aad9868","article-title":"Translational control by 5\u2032-untranslated regions of eukaryotic mRNAs","volume":"352","author":"Hinnebusch","year":"2016","journal-title":"Science"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"9689","DOI":"10.1093\/nar\/gkac739","article-title":"Global 5\u2032-UTR RNA structure regulates translation of a SERPINA1 mRNA","volume":"50","author":"Grayeski","year":"2022","journal-title":"Nucleic Acids Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"100","DOI":"10.1002\/jbmr.4156","article-title":"Multiple Endocrine Neoplasia Type 1 (MEN1) 5\u2032UTR Deletion, in MEN1 Family, Decreases Menin Expression","volume":"36","author":"Kooblall","year":"2021","journal-title":"J. Bone Miner. Res. Off. J. Am. Soc. Bone Miner. Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"e63021","DOI":"10.7554\/eLife.63021","article-title":"5\u2032-UTR SNP of FGF13 causes translational defect and intellectual disability","volume":"10","author":"Pan","year":"2021","journal-title":"ELife"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"11453","DOI":"10.1093\/nar\/gkad821","article-title":"The landscape of human SVA retrotransposons","volume":"51","author":"Chu","year":"2023","journal-title":"Nucleic Acids Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"11571","DOI":"10.1093\/nar\/gkae797","article-title":"G-quadruplexes in an SVA retrotransposon cause aberrant TAF1 gene expression in X-linked dystonia parkinsonism","volume":"52","author":"Nicoletto","year":"2024","journal-title":"Nucleic Acids Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"793","DOI":"10.1016\/j.ajhg.2020.04.011","article-title":"Expansion of GGC Repeat in GIPC1 Is Associated with Oculopharyngodistal Myopathy","volume":"106","author":"Deng","year":"2020","journal-title":"Am. J. Hum. Genet."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/2\/945\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,20]],"date-time":"2026-01-20T09:29:41Z","timestamp":1768901381000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/2\/945"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,18]]},"references-count":36,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["ijms27020945"],"URL":"https:\/\/doi.org\/10.3390\/ijms27020945","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,18]]}}}