{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,10]],"date-time":"2026-04-10T17:31:18Z","timestamp":1775842278682,"version":"3.50.1"},"reference-count":30,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2022,3,21]],"date-time":"2022-03-21T00:00:00Z","timestamp":1647820800000},"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. Med."],"abstract":"<jats:sec><jats:title>Background<\/jats:title><jats:p>The incorrect interpretation of missense and synonymous variants can lead to improper molecular diagnosis and subsequent faulty genetic counselling. The aim of this study was to evaluate the pathogenicity of presumed <jats:italic>COL4A3\/COL4A4<\/jats:italic> missense and synonymous variants detected by next-generation sequencing to provide evidence for diagnosis and genetic counselling.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Patients' clinical findings and genetic data were analysed retrospectively. An <jats:italic>in vitro<\/jats:italic> minigene assay was conducted to assess the effect of presumed <jats:italic>COL4A3\/COL4A4<\/jats:italic> missense and synonymous variants on RNA splicing.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Five unclassified <jats:italic>COL4A3\/COL4A4<\/jats:italic> variants, which were detected in five of 343 patients with hereditary kidney diseases, were analysed. All of them were predicted to affect splicing by Human Splicing Finder. The presumed <jats:italic>COL4A3<\/jats:italic> missense variant c.4793T &amp;gt; G [p. (Leu1598Arg)] resulted in a loss of alternative full-length transcript during the splicing process. The <jats:italic>COL4A3<\/jats:italic> transcript carried synonymous variant c.765G &amp;gt; A [p. (Thr255Thr)], led to an in-frame deletion of exon 13. Nevertheless, variants c.3566G &amp;gt; A [p. (Gly1189Glu)] in <jats:italic>COL4A3<\/jats:italic> and c.3990G &amp;gt; A [p. (Pro1330Pro)], c.4766C &amp;gt; T [p. (Pro1589Leu)] in <jats:italic>COL4A4<\/jats:italic> exhibited no deleterious effect on splicing. Among the five patients harbouring the abovementioned <jats:italic>COL4A3\/COL4A4<\/jats:italic> variants, three patients were genetically diagnosed with autosomal recessive Alport syndrome, one patient was highly suspected of having thin basement membrane nephropathy, and the other patient was clinically diagnosed with Alport syndrome.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p><jats:italic>COL4A3<\/jats:italic> presumed missense variant p. (Leu1598Arg) and synonymous variant p. (Thr255Thr) affect RNA splicing, which highlights the prime importance of transcript analysis of unclassified exonic sequence variants for better molecular diagnosis and genetic counselling. Meanwhile, the reliability of splicing predictions by predictive tools for exonic substitutions needs to be improved.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fmed.2022.838983","type":"journal-article","created":{"date-parts":[[2022,3,21]],"date-time":"2022-03-21T18:50:42Z","timestamp":1647888642000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":14,"title":["Presumed COL4A3\/COL4A4 Missense\/Synonymous Variants Induce Aberrant Splicing"],"prefix":"10.3389","volume":"9","author":[{"given":"Haiyue","family":"Deng","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yanqin","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jie","family":"Ding","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fang","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2022,3,21]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"224","DOI":"10.5858\/133.2.224","article-title":"Alport syndrome and thin glomerular basement membrane nephropathy: a practical approach to diagnosis","volume":"133","author":"Haas","year":"2009","journal-title":"Arch Pathol Lab Med."},{"key":"B2","doi-asserted-by":"publisher","first-page":"683","DOI":"10.1038\/nrg3051","article-title":"Understanding the contribution of synonymous mutations to human disease","volume":"12","author":"Sauna","year":"2011","journal-title":"Nat Rev Genet."},{"key":"B3","doi-asserted-by":"publisher","first-page":"1504","DOI":"10.1111\/liv.13754","article-title":"Presumed missense and synonymous mutations in ATP7B gene cause exon skipping in Wilson disease","volume":"38","author":"Wang","year":"2018","journal-title":"Liver Int."},{"key":"B4","doi-asserted-by":"publisher","first-page":"285","DOI":"10.1038\/nrg775","article-title":"Listening to silence and understanding nonsense: exonic mutations that affect splicing","volume":"3","author":"Cartegni","year":"2002","journal-title":"Nat Rev Genet."},{"key":"B5","doi-asserted-by":"publisher","first-page":"586","DOI":"10.1016\/j.jmoldx.2012.06.005","article-title":"Skin biopsy is a practical approach for the clinical diagnosis and molecular genetic analysis of X-linked Alport's syndrome","volume":"14","author":"Wang","year":"2012","journal-title":"J Mol Diagn."},{"key":"B6","doi-asserted-by":"publisher","first-page":"1532","DOI":"10.1002\/humu.22710","article-title":"Identification of splicing defects caused by mutations in the dysferlin gene","volume":"35","author":"Kergourlay","year":"2014","journal-title":"Hum Mutat."},{"key":"B7","doi-asserted-by":"publisher","first-page":"1268","DOI":"10.1111\/j.1523-1755.2005.00204.x","article-title":"Detection of mutations in the COL4A5 gene by analyzing cDNA of skin fibroblasts","volume":"67","author":"Wang","year":"2005","journal-title":"Kidney Int."},{"key":"B8","doi-asserted-by":"publisher","first-page":"97","DOI":"10.1146\/annurev-genom-083117-021641","article-title":"Rare-variant studies to complement genome-wide association studies","volume":"19","author":"Sazonovs","year":"2018","journal-title":"Annu Rev Genomics Hum Genet."},{"key":"B9","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":"2021","journal-title":"Kidney Int Rep."},{"key":"B10","doi-asserted-by":"publisher","first-page":"e0149241","DOI":"10.1371\/journal.pone.0149241","article-title":"Efficient targeted next generation sequencing-based workflow for differential diagnosis of alport-related disorders","volume":"11","author":"Kovacs","year":"2016","journal-title":"PLoS ONE."},{"key":"B11","doi-asserted-by":"publisher","first-page":"e1342","DOI":"10.1002\/mgg3.1342","article-title":"Pathogenic evaluation of synonymous COL4A5 variants in X-linked Alport syndrome using a minigene assay","volume":"8","author":"Horinouchi","year":"2020","journal-title":"Mol Genet Genomic Med."},{"key":"B12","doi-asserted-by":"publisher","first-page":"108","DOI":"10.1038\/nrm3742","article-title":"A day in the life of the spliceosome","volume":"15","author":"Matera","year":"2014","journal-title":"Nat Rev Mol Cell Biol."},{"key":"B13","doi-asserted-by":"publisher","first-page":"802","DOI":"10.1261\/rna.876308","article-title":"Splicing regulation: from a parts list of regulatory elements to an integrated splicing code","volume":"14","author":"Wang","year":"2008","journal-title":"RNA."},{"key":"B14","doi-asserted-by":"publisher","first-page":"386","DOI":"10.1038\/nrm1645","article-title":"Understanding alternative splicing: towards a cellular code","volume":"6","author":"Matlin","year":"2005","journal-title":"Nat Rev Mol Cell Biol."},{"key":"B15","doi-asserted-by":"publisher","first-page":"749","DOI":"10.1038\/nrg2164","article-title":"Splicing in disease: disruption of the splicing code and the decoding machinery","volume":"8","author":"Wang","year":"2007","journal-title":"Nat Rev Genet."},{"key":"B16","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":"B17","doi-asserted-by":"publisher","first-page":"22225","DOI":"10.1038\/s41598-021-01604-9","article-title":"Author Correction: 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":"B18","doi-asserted-by":"publisher","first-page":"306","DOI":"10.1111\/hae.13687","article-title":"Splicing analysis of 26 F8 nucleotide variations using a minigene assay","volume":"25","author":"Jourdy","year":"2019","journal-title":"Haemophilia."},{"key":"B19","doi-asserted-by":"publisher","first-page":"e1451","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":"B20","doi-asserted-by":"publisher","first-page":"1228","DOI":"10.1002\/humu.22101","article-title":"Guidelines for splicing analysis in molecular diagnosis derived from a set of 327 combined in silico\/in vitro studies on BRCA1 and BRCA2 variants","volume":"33","author":"Houdayer","year":"2012","journal-title":"Hum Mutat."},{"key":"B21","doi-asserted-by":"publisher","first-page":"810","DOI":"10.1038\/embor.2009.170","article-title":"Missed threads. The impact of pre-mRNA splicing defects on clinical practice","volume":"10","author":"Baralle","year":"2009","journal-title":"EMBO Rep."},{"key":"B22","doi-asserted-by":"publisher","first-page":"331","DOI":"10.1016\/j.ymeth.2005.07.015","article-title":"Use of minigene systems to dissect alternative splicing elements","volume":"37","author":"Cooper","year":"2005","journal-title":"Methods."},{"key":"B23","doi-asserted-by":"publisher","first-page":"438","DOI":"10.1136\/jmg.2007.056895","article-title":"Screening BRCA1 and BRCA2 unclassified variants for splicing mutations using reverse transcription PCR on patient RNA and an ex vivo assay based on a splicing reporter minigene","volume":"45","author":"Bonnet","year":"2008","journal-title":"J Med Genet."},{"key":"B24","doi-asserted-by":"publisher","first-page":"398","DOI":"10.1136\/jmg.2009.074047","article-title":"The BRCA1 c.5434C->G (p.Pro1812Ala) variant induces a deleterious exon 23 skipping by affecting exonic splicing regulatory elements","volume":"47","author":"Gaildrat","year":"2010","journal-title":"J Med Genet."},{"key":"B25","doi-asserted-by":"publisher","first-page":"327","DOI":"10.1002\/mgg3.145","article-title":"Splicing analysis for exonic and intronic mismatch repair gene variants associated with Lynch syndrome confirms high concordance between minigene assays and patient RNA analyses","volume":"3","author":"van der Klift","year":"2015","journal-title":"Mol Genet Genomic Med."},{"key":"B26","doi-asserted-by":"publisher","first-page":"1412","DOI":"10.1002\/humu.20796","article-title":"A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects","volume":"29","author":"Tournier","year":"2008","journal-title":"Hum Mutat."},{"key":"B27","doi-asserted-by":"publisher","first-page":"2342","DOI":"10.1016\/S0021-9258(17)42173-9","article-title":"Alternative splicing of the NC1 domain of the human alpha 3(IV) collagen gene. Differential expression of mRNA transcripts that predict three protein variants with distinct carboxyl regions","volume":"269","author":"Feng","year":"1994","journal-title":"J Biol Chem."},{"key":"B28","doi-asserted-by":"publisher","first-page":"1535","DOI":"10.1007\/s00467-014-2797-4","article-title":"Natural history of genetically proven autosomal recessive Alport syndrome","volume":"29","author":"Oka","year":"2014","journal-title":"Pediatr Nephrol."},{"key":"B29","doi-asserted-by":"publisher","first-page":"357","DOI":"10.1016\/j.jacl.2017.01.013","article-title":"A novel frameshift mutation in the lipoprotein lipase gene is rescued by alternative messenger RNA splicing","volume":"11","author":"Laurie","year":"2017","journal-title":"J Clin Lipidol."},{"key":"B30","doi-asserted-by":"publisher","first-page":"680","DOI":"10.1126\/science.8430317","article-title":"The skipping of constitutive exons in vivo induced by nonsense mutations","volume":"259","author":"Dietz","year":"1993","journal-title":"Science."}],"container-title":["Frontiers in Medicine"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmed.2022.838983\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,3,21]],"date-time":"2022-03-21T18:50:44Z","timestamp":1647888644000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmed.2022.838983\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,3,21]]},"references-count":30,"alternative-id":["10.3389\/fmed.2022.838983"],"URL":"https:\/\/doi.org\/10.3389\/fmed.2022.838983","relation":{},"ISSN":["2296-858X"],"issn-type":[{"value":"2296-858X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,3,21]]},"article-number":"838983"}}