{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,7,30]],"date-time":"2025-07-30T15:54:34Z","timestamp":1753890874939,"version":"3.41.2"},"reference-count":37,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,6,11]],"date-time":"2025-06-11T00:00:00Z","timestamp":1749600000000},"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:sec><jats:title>Introduction<\/jats:title><jats:p>X-linked Alport syndrome (XLAS), caused by mutations in the COL4A5 gene, is an X-linked hereditary disease typically characterized by renal failure, hearing loss, and ocular abnormalities. It is a leading hereditary cause of end-stage renal disease (ESRD) worldwide. Studies on the genotype-phenotype correlation in Alport syndrome suggest that splicing mutations result in more severe clinical phenotypes than missense mutations. Determining whether COL4A5 mutations lead to aberrant mRNA splicing is critical for diagnosis and prognosis.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>This study retrospectively reviewed pediatric XLAS patients with COL4A5 gene mutations from a single-center cohort, summarizing and analyzing their clinical features. Minigene assay was employed to evaluate the mRNA splicing functionality of 26 single-nucleotide variants (SNVs), both intronic and exonic, identified in XLAS patients. Bioinformatics tools were used to evaluate the accuracy and sensitivity of splicing mutation prediction. Additionally, linear mixed models were applied to analyze the relationship between mutation types and prognosis in patients\u2019 estimated glomerular filtration rate (eGFR), exploring genotype-phenotype correlations.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>In this cohort, we screened 41 XLAS pediatric patients, including 32 with confirmed XLAS and nine suspected XLAS. The cohort included 21 males (51.2%) and 20 females (48.8%), with a median age at onset of 4.42 years. Among the patients, 22 presented with both hematuria and proteinuria, while 18 exhibited hematuria alone. Notably, only one patient had isolated proteinuria. Regarding mRNA splicing, among the 26 intronic and exonic SNVs, 10 mutations (38.5%) were found to cause aberrant mRNA splicing, as demonstrated by the minigene assay. Sensitivity and specificity assessments of bioinformatics tools revealed that ESE Finder demonstrated higher sensitivity, while RNA Splicer exhibited greater specificity. Furthermore, These splicing abnormalities were closely associated with a faster decline in eGFR.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>This study demonstrates that 38.5% of SNVs in the COL4A5 gene result in aberrant mRNA splicing, which is closely linked to renal function decline in XLAS. Splicing mutations are correlated with more rapid renal progression, highlighting the importance of determining the splicing effects of SNVs during genetic screening for XLAS.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fgene.2025.1564343","type":"journal-article","created":{"date-parts":[[2025,6,11]],"date-time":"2025-06-11T05:14:13Z","timestamp":1749618853000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["A comprehensive splicing characterization of COL4A5 mutations and prognostic significance in a single cohort with X-linked alport syndrome"],"prefix":"10.3389","volume":"16","author":[{"given":"Haomiao","family":"Li","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shengnan","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wei","family":"Zhou","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chunli","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chunhua","family":"Zhu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sanlong","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fei","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhanjun","family":"Jia","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aihua","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bixia","family":"Zheng","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Guixia","family":"Ding","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2025,6,11]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"108","DOI":"10.1016\/j.ekir.2021.10.012","article-title":"Last nucleotide substitutions of COL4A5 exons cause aberrant splicing","volume":"7","author":"Aoto","year":"2022","journal-title":"Kidney Int. Rep."},{"key":"B2","doi-asserted-by":"publisher","first-page":"367","DOI":"10.1016\/j.kint.2023.05.006","article-title":"A wave of deep intronic mutations in X-linked Alport syndrome","volume":"104","author":"Boisson","year":"2023","journal-title":"Kidney Int."},{"key":"B3","doi-asserted-by":"publisher","first-page":"1705","DOI":"10.1002\/humu.24074","article-title":"Variant effect on splicing regulatory elements, branchpoint usage, and pseudoexonization: strategies to enhance bioinformatic prediction using hereditary cancer genes as exemplars","volume":"41","author":"Canson","year":"2020","journal-title":"Hum. Mutat."},{"key":"B4","doi-asserted-by":"publisher","first-page":"13","DOI":"10.1186\/s13059-016-0881-8","article-title":"A survey of best practices for RNA-seq data analysis","volume":"17","author":"Conesa","year":"2016","journal-title":"Genome Biol."},{"key":"B5","doi-asserted-by":"publisher","first-page":"302","DOI":"10.1002\/humu.23364","article-title":"Urine-derived podocytes-lineage cells: a promising tool for precision medicine in Alport Syndrome","volume":"39","author":"Daga","year":"2018","journal-title":"Hum. Mutat."},{"key":"B6","doi-asserted-by":"publisher","first-page":"480","DOI":"10.1038\/s41431-019-0537-8","article-title":"New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene editing in podocyte-lineage cells","volume":"28","author":"Daga","year":"2020","journal-title":"Eur. J. Hum. Genet."},{"key":"B7","doi-asserted-by":"publisher","first-page":"100898","DOI":"10.1016\/j.preteyeres.2020.100898","article-title":"The X-linked retinopathies: physiological insights, pathogenic mechanisms, phenotypic features and novel therapies","volume":"82","author":"De Silva","year":"2021","journal-title":"Prog. Retin. Eye Res."},{"key":"B8","doi-asserted-by":"publisher","first-page":"2487","DOI":"10.1093\/ndt\/gfac002","article-title":"Dissecting the genotype-phenotype correlation of COL4A5 gene mutation and its response to renin-angiotensin-aldosterone system blockers in Chinese male patients with Alport syndrome","volume":"37","author":"Di","year":"2022","journal-title":"Nephrol. Dial. Transpl."},{"key":"B9","doi-asserted-by":"publisher","first-page":"689","DOI":"10.1038\/nrg3778","article-title":"Context-dependent control of alternative splicing by RNA-binding proteins","volume":"15","author":"Fu","year":"2014","journal-title":"Nat. Rev. Genet."},{"key":"B10","doi-asserted-by":"publisher","first-page":"2756","DOI":"10.1038\/s41467-021-22780-2","article-title":"Hotspot exons are common targets of splicing perturbations","volume":"12","author":"Glidden","year":"2021","journal-title":"Nat. Commun."},{"key":"B11","doi-asserted-by":"publisher","first-page":"142","DOI":"10.1056\/NEJMoa1806891","article-title":"Diagnostic utility of exome sequencing for kidney disease","volume":"380","author":"Groopman","year":"2019","journal-title":"N. Engl. J. Med."},{"key":"B12","doi-asserted-by":"publisher","first-page":"854","DOI":"10.1038\/s42003-023-05203-4","article-title":"iPSC-derived type IV collagen \u03b15-expressing kidney organoids model Alport syndrome","volume":"6","author":"Hirayama","year":"2023","journal-title":"Commun. Biol."},{"key":"B13","doi-asserted-by":"publisher","first-page":"2543","DOI":"10.1056\/NEJMra022296","article-title":"Alport\u2019s syndrome, Goodpasture\u2019s syndrome, and type IV collagen","volume":"348","author":"Hudson","year":"2003","journal-title":"N. Engl. J. Med."},{"key":"B14","doi-asserted-by":"publisher","first-page":"649","DOI":"10.1681\/ASN.V114649","article-title":"X-linked Alport syndrome: natural history in 195 families and genotype-phenotype correlations in males","volume":"11","author":"Jais","year":"2000","journal-title":"J. Am. Soc. Nephrol. JASN"},{"key":"B15","doi-asserted-by":"publisher","first-page":"718","DOI":"10.1016\/j.ekir.2020.01.008","article-title":"Trimerization and genotype-phenotype correlation of COL4A5 mutants in Alport syndrome","volume":"5","author":"Kamura","year":"2020","journal-title":"Kidney Int. Rep."},{"key":"B16","doi-asserted-by":"publisher","first-page":"272","DOI":"10.1053\/j.ajkd.2020.03.026","article-title":"Alport syndrome: achieving early diagnosis and treatment","volume":"77","author":"Kashtan","year":"2021","journal-title":"Am. J. Kidney Dis."},{"key":"B17","doi-asserted-by":"publisher","first-page":"323","DOI":"10.1186\/1471-2105-12-323","article-title":"RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome","volume":"12","author":"Li","year":"2011","journal-title":"BMC Bioinforma."},{"key":"B18","doi-asserted-by":"publisher","first-page":"1489","DOI":"10.1016\/S0140-6736(17)31611-2","article-title":"Ataluren in patients with nonsense mutation Duchenne muscular dystrophy (ACT DMD): a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial","volume":"390","author":"McDonald","year":"2017","journal-title":"Lancet"},{"key":"B19","doi-asserted-by":"publisher","first-page":"2740","DOI":"10.1681\/ASN.2013080912","article-title":"Improving mutation screening in familial hematuric nephropathies through next generation sequencing","volume":"25","author":"Morini\u00e8re","year":"2014","journal-title":"J. Am. Soc. Nephrol."},{"key":"B20","doi-asserted-by":"publisher","first-page":"158","DOI":"10.1007\/s10157-018-1629-4","article-title":"A review of clinical characteristics and genetic backgrounds in Alport syndrome","volume":"23","author":"Nozu","year":"2019","journal-title":"Clin. Exp. Nephrol."},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.1002\/wrna.1451","article-title":"Rules and tools to predict the splicing effects of exonic and intronic mutations","volume":"9","author":"Ohno","year":"2018","journal-title":"Wiley Interdiscip. Rev. RNA"},{"key":"B22","doi-asserted-by":"publisher","first-page":"367","DOI":"10.2471\/BLT.13.128728","article-title":"The global burden of disabling hearing impairment: a call to action","volume":"92","author":"Olusanya","year":"2014","journal-title":"Bull. World Health Organ."},{"key":"B23","doi-asserted-by":"publisher","first-page":"103891","DOI":"10.1016\/j.isci.2022.103891","article-title":"NanoLuc reporters identify COL4A5 nonsense mutations susceptible to drug-induced stop codon readthrough","volume":"25","author":"Omachi","year":"2022","journal-title":"iScience"},{"key":"B24","doi-asserted-by":"publisher","first-page":"5154","DOI":"10.3390\/ijms22105154","article-title":"Principles and practical considerations for the analysis of disease-associated alternative splicing events using the gateway cloning-based minigene vectors pDESTsplice and pSpliceExpress","volume":"22","author":"Putscher","year":"2021","journal-title":"Int. J. Mol. Sci."},{"key":"B25","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":"B26","doi-asserted-by":"publisher","first-page":"511","DOI":"10.1007\/s40259-024-00665-2","article-title":"Mechanisms of action of the US food and drug administration-approved antisense oligonucleotide drugs","volume":"38","author":"Sang","year":"2024","journal-title":"BioDrugs"},{"key":"B27","doi-asserted-by":"publisher","first-page":"1175","DOI":"10.1007\/s00467-018-3985-4","article-title":"Expert consensus guidelines for the genetic diagnosis of Alport syndrome","volume":"34","author":"Savige","year":"2019","journal-title":"Pediatr. Nephrol."},{"key":"B28","doi-asserted-by":"publisher","first-page":"143","DOI":"10.2215\/CJN.04230321","article-title":"Guidelines for genetic testing and management of Alport syndrome","volume":"17","author":"Savige","year":"2022","journal-title":"Clin. J. Am. Soc. Nephrol."},{"key":"B29","doi-asserted-by":"publisher","first-page":"703","DOI":"10.2215\/CJN.10581014","article-title":"Ocular features in Alport syndrome: pathogenesis and clinical significance","volume":"10","author":"Savige","year":"2015","journal-title":"Clin. J. Am. Soc. Nephrol."},{"key":"B30","doi-asserted-by":"publisher","first-page":"701","DOI":"10.1016\/j.cell.2009.02.009","article-title":"The spliceosome: design principles of a dynamic RNP machine","volume":"136","author":"Wahl","year":"2009","journal-title":"Cell."},{"key":"B31","doi-asserted-by":"publisher","first-page":"18097","DOI":"10.1038\/s41598-021-97414-0","article-title":"mRNA analysis identifies deep intronic variants causing Alport syndrome and overcomes the problem of negative results of exome sequencing","volume":"11","author":"Wang","year":"2021","journal-title":"Sci. Rep."},{"key":"B32","doi-asserted-by":"publisher","first-page":"1118","DOI":"10.1111\/gtc.13170","article-title":"Investigation of exon skipping therapy in kidney organoids from Alport syndrome patients derived iPSCs","volume":"29","author":"Yabuuchi","year":"2024","journal-title":"Genes. Cells Devoted Mol. Cell. Mech."},{"key":"B33","doi-asserted-by":"publisher","first-page":"2777","DOI":"10.1038\/s41467-020-16605-x","article-title":"Development of an exon skipping therapy for X-linked Alport syndrome with truncating variants in COL4A5","volume":"11","author":"Yamamura","year":"","journal-title":"Nat. Commun."},{"key":"B34","doi-asserted-by":"publisher","first-page":"1605","DOI":"10.1016\/j.kint.2020.06.038","article-title":"Genotype-phenotype correlations influence the response to angiotensin-targeting drugs in Japanese patients with male X-linked Alport syndrome","volume":"98","author":"Yamamura","year":"","journal-title":"Kidney Int."},{"key":"B35","doi-asserted-by":"publisher","first-page":"320","DOI":"10.1053\/j.ajkd.2024.02.005","article-title":"Clinical significance of the cystic phenotype in Alport syndrome","volume":"84","author":"Zeni","year":"2024","journal-title":"Am. J. Kidney Dis."},{"key":"B36","doi-asserted-by":"publisher","first-page":"129","DOI":"10.2478\/jtim-2024-0005","article-title":"Progress in therapeutic targets on podocyte for Alport syndrome","volume":"12","author":"Zheng","year":"2024","journal-title":"J. Transl. Intern. Med."},{"key":"B37","doi-asserted-by":"publisher","first-page":"592611","DOI":"10.3389\/fgene.2020.592611","article-title":"A comprehensive analysis and splicing characterization of naturally occurring synonymous variants in the ATP7B gene","volume":"11","author":"Zhou","year":"2020","journal-title":"Front. Genet."}],"container-title":["Frontiers in Genetics"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fgene.2025.1564343\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,6,11]],"date-time":"2025-06-11T05:14:15Z","timestamp":1749618855000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fgene.2025.1564343\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,6,11]]},"references-count":37,"alternative-id":["10.3389\/fgene.2025.1564343"],"URL":"https:\/\/doi.org\/10.3389\/fgene.2025.1564343","relation":{},"ISSN":["1664-8021"],"issn-type":[{"type":"electronic","value":"1664-8021"}],"subject":[],"published":{"date-parts":[[2025,6,11]]},"article-number":"1564343"}}