mRNA analysis identifies deep intronic variants causing Alport syndrome and overcomes the problem of negative results of exome sequencing

Abstract Mutations in COL4A3, COL4A4 and COL4A5 genes lead to Alport syndrome (AS). 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 NGS test results....

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Autores principales:	Xiaoyuan Wang, Yanqin Zhang, Jie Ding, Fang Wang
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/e1eb685e39b8474eb28b05495b941994
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spelling	oai:doaj.org-article:e1eb685e39b8474eb28b05495b9419942021-12-02T17:19:16ZmRNA analysis identifies deep intronic variants causing Alport syndrome and overcomes the problem of negative results of exome sequencing10.1038/s41598-021-97414-02045-2322https://doaj.org/article/e1eb685e39b8474eb28b05495b9419942021-09-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-97414-0https://doaj.org/toc/2045-2322Abstract Mutations in COL4A3, COL4A4 and COL4A5 genes lead to Alport syndrome (AS). 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 NGS test results. Urine COL4A3–5 mRNAs were analyzed in the probands with an uncertain inherited mode of AS, and COL4A5 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 COL4A4 and COL4A5 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, COL4A5 variants c.2677 + 646 C > T and r.2678_r.2767del were detected at the DNA and RNA level, respectively, whereas it is unclear whether c.2677 + 646 C > T 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 COL4A3–5 mRNA from urine as the first choice for these patients because it is feasible and non-invasive.Xiaoyuan WangYanqin ZhangJie DingFang WangNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-13 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Xiaoyuan Wang Yanqin Zhang Jie Ding Fang Wang mRNA analysis identifies deep intronic variants causing Alport syndrome and overcomes the problem of negative results of exome sequencing
description	Abstract Mutations in COL4A3, COL4A4 and COL4A5 genes lead to Alport syndrome (AS). 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 NGS test results. Urine COL4A3–5 mRNAs were analyzed in the probands with an uncertain inherited mode of AS, and COL4A5 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 COL4A4 and COL4A5 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, COL4A5 variants c.2677 + 646 C > T and r.2678_r.2767del were detected at the DNA and RNA level, respectively, whereas it is unclear whether c.2677 + 646 C > T 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 COL4A3–5 mRNA from urine as the first choice for these patients because it is feasible and non-invasive.
format	article
author	Xiaoyuan Wang Yanqin Zhang Jie Ding Fang Wang
author_facet	Xiaoyuan Wang Yanqin Zhang Jie Ding Fang Wang
author_sort	Xiaoyuan Wang
title	mRNA analysis identifies deep intronic variants causing Alport syndrome and overcomes the problem of negative results of exome sequencing
title_short	mRNA analysis identifies deep intronic variants causing Alport syndrome and overcomes the problem of negative results of exome sequencing
title_full	mRNA analysis identifies deep intronic variants causing Alport syndrome and overcomes the problem of negative results of exome sequencing
title_fullStr	mRNA analysis identifies deep intronic variants causing Alport syndrome and overcomes the problem of negative results of exome sequencing
title_full_unstemmed	mRNA analysis identifies deep intronic variants causing Alport syndrome and overcomes the problem of negative results of exome sequencing
title_sort	mrna analysis identifies deep intronic variants causing alport syndrome and overcomes the problem of negative results of exome sequencing
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/e1eb685e39b8474eb28b05495b941994
work_keys_str_mv	AT xiaoyuanwang mrnaanalysisidentifiesdeepintronicvariantscausingalportsyndromeandovercomestheproblemofnegativeresultsofexomesequencing AT yanqinzhang mrnaanalysisidentifiesdeepintronicvariantscausingalportsyndromeandovercomestheproblemofnegativeresultsofexomesequencing AT jieding mrnaanalysisidentifiesdeepintronicvariantscausingalportsyndromeandovercomestheproblemofnegativeresultsofexomesequencing AT fangwang mrnaanalysisidentifiesdeepintronicvariantscausingalportsyndromeandovercomestheproblemofnegativeresultsofexomesequencing
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mRNA analysis identifies deep intronic variants causing Alport syndrome and overcomes the problem of negative results of exome sequencing

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