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However, pathogenic variants in some AS patients are not detected by exome sequencing. The aim of this study was to identify the underlying genetic causes of five unrelated AS probands with negative next-generation sequencing (NGS) test results. Urine <jats:italic>COL4A3\u20135<\/jats:italic> mRNAs were analyzed in the probands with an uncertain inherited mode of AS, and <jats:italic>COL4A5<\/jats:italic> mRNA of skin fibroblasts was analyzed in the probands with X-linked AS. RT-PCR and direct sequencing were performed to detect mRNA abnormalities. PCR and direct sequencing were used to analyze the exons with flanking intronic sequences corresponding to mRNA abnormalities. Six novel deep intronic splicing variants in <jats:italic>COL4A4<\/jats:italic> and <jats:italic>COL4A5<\/jats:italic> genes that cannot be captured by exome sequencing were identified in the four AS probands. Skipping of an exon was caused by an intronic variant, and retention of an intron fragment caused by five variants. In the remaining AS proband, <jats:italic>COL4A5<\/jats:italic> variants c.2677\u2009+\u2009646 C\u2009&gt;\u2009T and r.2678_r.2767del were detected at the DNA and RNA level, respectively, whereas it is unclear whether c.2677\u2009+\u2009646 C\u2009&gt;\u2009T may not lead to r.2678_r.2767del. Our results reveal that mRNA analysis for AS genes from either urine or skin fibroblasts can resolve genetic diagnosis in AS patients with negative NGS results. We recommend analyzing <jats:italic>COL4A3\u20135<\/jats:italic> mRNA from urine as the first choice for these patients because it is feasible and non-invasive.<\/jats:p>","DOI":"10.1038\/s41598-021-97414-0","type":"journal-article","created":{"date-parts":[[2021,9,10]],"date-time":"2021-09-10T10:02:45Z","timestamp":1631268165000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":26,"title":["mRNA analysis identifies deep intronic variants causing Alport syndrome and overcomes the problem of negative results of exome sequencing"],"prefix":"10.1038","volume":"11","author":[{"given":"Xiaoyuan","family":"Wang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanqin","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jie","family":"Ding","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fang","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,9,10]]},"reference":[{"key":"97414_CR1","doi-asserted-by":"publisher","first-page":"170","DOI":"10.1038\/nrneph.2012.259","volume":"9","author":"J Kruegel","year":"2013","unstructured":"Kruegel, J., Rubel, D. & Gross, O. 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