Nanopore Deep Sequencing as a Tool to Characterize and Quantify Aberrant Splicing Caused by Variants in Inherited Retinal Dystrophy Genes.

Publisher: MDPI Country of Publication: Switzerland NLM ID: 101092791 Publication Model: Electronic Cited Medium: Internet ISSN: 1422-0067 (Electronic) Linking ISSN: 14220067 NLM ISO Abbreviation: Int J Mol Sci Subsets: MEDLINE

Original Publication: Basel, Switzerland : MDPI, [2000-

Retinal Dystrophies*/genetics ; Retinal Dystrophies*/diagnosis ; Nanopore Sequencing*/methods ; High-Throughput Nucleotide Sequencing*/methods; Humans ; Alternative Splicing/genetics ; RNA Splicing/genetics ; Exons/genetics

The contribution of splicing variants to molecular diagnostics of inherited diseases is reported to be less than 10%. This figure is likely an underestimation due to several factors including difficulty in predicting the effect of such variants, the need for functional assays, and the inability to detect them (depending on their locations and the sequencing technology used). The aim of this study was to assess the utility of Nanopore sequencing in characterizing and quantifying aberrant splicing events. For this purpose, we selected 19 candidate splicing variants that were identified in patients affected by inherited retinal dystrophies. Several in silico tools were deployed to predict the nature and estimate the magnitude of variant-induced aberrant splicing events. Minigene assay or whole blood-derived cDNA was used to functionally characterize the variants. PCR amplification of minigene-specific cDNA or the target gene in blood cDNA, combined with Nanopore sequencing, was used to identify the resulting transcripts. Thirteen out of nineteen variants caused aberrant splicing events, including cryptic splice site activation, exon skipping, pseudoexon inclusion, or a combination of these. Nanopore sequencing allowed for the identification of full-length transcripts and their precise quantification, which were often in accord with in silico predictions. The method detected reliably low-abundant transcripts, which would not be detected by conventional strategies, such as RT-PCR followed by Sanger sequencing.

Int J Mol Sci. 2021 May 20;22(10):. (PMID: 34065499)
Sci Rep. 2018 Sep 6;8(1):13312. (PMID: 30190494)
Cells. 2022 Nov 17;11(22):. (PMID: 36429068)
Am J Ophthalmol Case Rep. 2022 Sep 06;28:101699. (PMID: 36118280)
Am J Hum Genet. 2023 Jul 6;110(7):1046-1067. (PMID: 37352859)
Genet Med. 2019 Aug;21(8):1751-1760. (PMID: 30643219)
Genet Med. 2019 Aug;21(8):1761-1771. (PMID: 30670881)
Hum Genet. 2014 Jan;133(1):1-9. (PMID: 24077912)
Hum Mutat. 2021 Jul;42(7):799-810. (PMID: 33942434)
JAMA Ophthalmol. 2021 Jul 01;139(7):691-700. (PMID: 34014271)
Prog Retin Eye Res. 2010 Sep;29(5):335-75. (PMID: 20362068)
Int J Mol Sci. 2021 Feb 03;22(4):. (PMID: 33546218)
Hum Mutat. 2015 Jan;36(1):43-7. (PMID: 25363634)
Hum Mutat. 2020 Jan;41(1):255-264. (PMID: 31544997)
Int J Mol Sci. 2024 Jun 13;25(12):. (PMID: 38928247)
Am J Ophthalmol. 2023 May;249:57-73. (PMID: 36592879)
Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W187-90. (PMID: 15215377)
Bioinformatics. 2022 Aug 2;38(15):3741-3748. (PMID: 35639973)
Nucleic Acids Res. 1987 Sep 11;15(17):7155-74. (PMID: 3658675)
Int J Mol Sci. 2021 Jan 16;22(2):. (PMID: 33467000)
Int J Mol Sci. 2022 Mar 31;23(7):. (PMID: 35409265)
J Comput Biol. 2004;11(2-3):377-94. (PMID: 15285897)
Cold Spring Harb Mol Case Stud. 2018 Aug 1;4(4):. (PMID: 29848554)
Int J Mol Sci. 2018 Jul 27;19(8):. (PMID: 30060493)
Genet Med. 2020 Jul;22(7):1235-1246. (PMID: 32307445)
Hum Mutat. 2015 Jan;36(1):39-42. (PMID: 25346251)
Int J Genomics. 2022 Oct 13;2022:5265686. (PMID: 36275637)
Genome Res. 2018 Jan;28(1):100-110. (PMID: 29162642)
NPJ Genom Med. 2024 Jan 20;9(1):6. (PMID: 38245557)
Cell. 2019 Jan 24;176(3):535-548.e24. (PMID: 30661751)
Nucleic Acids Res. 2003 Jul 1;31(13):3568-71. (PMID: 12824367)
Hum Mol Genet. 2017 Aug 1;26(R1):R2-R11. (PMID: 28510639)
Sci Rep. 2022 Dec 2;12(1):20815. (PMID: 36460718)
Prog Retin Eye Res. 2020 Nov;79:100861. (PMID: 32278709)
Audiol Neurootol. 2011;16(2):93-105. (PMID: 21252500)
Genome Biol. 2022 Apr 21;23(1):103. (PMID: 35449021)
Nucleic Acids Res. 2001 Mar 1;29(5):1185-90. (PMID: 11222768)
Methods Mol Biol. 2010;653:249-57. (PMID: 20721748)
J Comput Biol. 1997 Fall;4(3):311-23. (PMID: 9278062)
Nucleic Acids Res. 2018 Sep 6;46(15):7913-7923. (PMID: 29750258)
Exp Eye Res. 2022 Dec;225:109276. (PMID: 36209838)
Hum Mol Genet. 2013 Dec 20;22(25):5136-45. (PMID: 23918662)
Genes (Basel). 2021 Jan 06;12(1):. (PMID: 33418956)
J Med Genet. 2024 Jan 19;61(2):186-195. (PMID: 37734845)
Genet Med. 2020 Jun;22(6):1005-1014. (PMID: 32123317)
Am J Hum Genet. 2017 Jan 5;100(1):75-90. (PMID: 28041643)
Bioinformatics. 2009 Aug 15;25(16):2078-9. (PMID: 19505943)
Hum Mol Genet. 2014 Dec 20;23(25):6797-806. (PMID: 25082829)
Genes (Basel). 2023 Apr 18;14(4):. (PMID: 37107692)
Bioinformatics. 2018 Sep 15;34(18):3094-3100. (PMID: 29750242)
Proc Natl Acad Sci U S A. 2020 Feb 4;117(5):2710-2716. (PMID: 31964843)
Hum Mutat. 2011 Apr;32(4):436-44. (PMID: 21309043)
Prog Retin Eye Res. 2021 Jan;80:100874. (PMID: 32553897)
Invest Ophthalmol Vis Sci. 2011 Oct 31;52(11):8479-87. (PMID: 21911583)

1371 Velux Stiftung

Keywords: Nanopore; exon skipping; inherited retinal dystrophies (IRDs); long-read sequencing; minigene assay; pseudoexon; splice variant; splicing

Date Created: 20240914 Date Completed: 20240914 Latest Revision: 20240916

20250114

PMC11395040

10.3390/ijms25179569

39273516