دورية أكاديمية
أعرض في EDS

Minigene Splicing Assays and Long-Read Sequencing to Unravel Pathogenic Deep-Intronic Variants in PAX6 in Congenital Aniridia.

التفاصيل البيبلوغرافية
العنوان: Minigene Splicing Assays and Long-Read Sequencing to Unravel Pathogenic Deep-Intronic Variants in PAX6 in Congenital Aniridia.
المؤلفون: Tamayo A; Department of Genetics & Genomics, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), 28040 Madrid, Spain.; Center for Biomedical Network Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, 28029 Madrid, Spain.; Department of Surgery, Medical and Social Sciences, Faculty of Medicine and Health Sciences, Science and Technology Campus, University of Alcalá, 28871 Alcalá de Henares, Spain., Núñez-Moreno G; Department of Genetics & Genomics, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), 28040 Madrid, Spain.; Center for Biomedical Network Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, 28029 Madrid, Spain.; Bioinformatics Unit, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), 28240 Madrid, Spain., Ruiz C; Department of Genetics & Genomics, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), 28040 Madrid, Spain., Plaisancie J; Centre de Référence des Affections Rares en Génétique Ophtalmologique (CARGO), Hôpital Purpan, CHU Toulouse, 31000 Toulouse, France.; INSERM U1214, Université Toulouse III, 31000 Toulouse, France., Damian A; Department of Genetics & Genomics, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), 28040 Madrid, Spain.; Center for Biomedical Network Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, 28029 Madrid, Spain., Moya J; Department of Genetics & Genomics, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), 28040 Madrid, Spain., Chassaing N; Centre de Référence des Affections Rares en Génétique Ophtalmologique (CARGO), Hôpital Purpan, CHU Toulouse, 31000 Toulouse, France.; INSERM U1214, Université Toulouse III, 31000 Toulouse, France., Calvas P; Centre de Référence des Affections Rares en Génétique Ophtalmologique (CARGO), Hôpital Purpan, CHU Toulouse, 31000 Toulouse, France.; INSERM U1214, Université Toulouse III, 31000 Toulouse, France., Ayuso C; Department of Genetics & Genomics, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), 28040 Madrid, Spain.; Center for Biomedical Network Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, 28029 Madrid, Spain., Minguez P; Department of Genetics & Genomics, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), 28040 Madrid, Spain.; Center for Biomedical Network Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, 28029 Madrid, Spain.; Bioinformatics Unit, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), 28240 Madrid, Spain., Corton M; Department of Genetics & Genomics, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), 28040 Madrid, Spain.; Center for Biomedical Network Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, 28029 Madrid, Spain.
المصدر: International journal of molecular sciences [Int J Mol Sci] 2023 Jan 13; Vol. 24 (2). Date of Electronic Publication: 2023 Jan 13.
نوع المنشور: Journal Article
اللغة: English
بيانات الدورية: 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
أسماء مطبوعة: Original Publication: Basel, Switzerland : MDPI, [2000-
مواضيع طبية MeSH: Aniridia*/genetics , RNA Splicing*/genetics, Humans ; Alternative Splicing/genetics ; Introns/genetics ; Mutation ; PAX6 Transcription Factor/genetics ; PAX6 Transcription Factor/metabolism ; RNA Splice Sites
مستخلص: PAX6 haploinsufficiency causes aniridia, a congenital eye disorder that involves the iris, and foveal hypoplasia. Comprehensive screening of the PAX6 locus, including the non-coding regions, by next-generation sequencing revealed four deep-intronic variants with potential effects on pre-RNA splicing. Nevertheless, without a functional analysis, their pathogenicity could not be established. We aimed to decipher their impact on the canonical PAX6 splicing using in vitro minigene splicing assays and nanopore-based long-read sequencing. Two multi-exonic PAX6 constructs were generated, and minigene assays were carried out. An aberrant splicing pattern was observed for two variants in intron 6, c.357+136G>A and c.357+334G>A. In both cases, several exonization events, such as pseudoexon inclusions and partial intronic retention, were observed due to the creation or activation of new/cryptic non-canonical splicing sites, including a shared intronic donor site. In contrast, two variants identified in intron 11, c.1032+170A>T and c.1033-275A>C, seemed not to affect splicing processes. We confirmed the high complexity of alternative splicing of PAX6 exon 6, which also involves unreported cryptic intronic sites. Our study highlights the importance of integrating functional studies into diagnostic algorithms to decipher the potential implication of non-coding variants, usually classified as variants of unknown significance, thus allowing variant reclassification to achieve a conclusive genetic diagnosis.
References: Prog Retin Eye Res. 2022 Oct 21;:101133. (PMID: 36280537)
Gene. 2017 Sep 30;630:44-48. (PMID: 28760551)
Genes (Basel). 2019 Dec 17;10(12):. (PMID: 31861090)
Folia Biol (Praha). 2020;66(4):123-132. (PMID: 33745259)
Hum Genet. 2018 Oct;137(10):831-846. (PMID: 30291432)
J Hum Genet. 2021 Mar;66(3):333-338. (PMID: 32920601)
Genome Res. 2018 Jan;28(1):100-110. (PMID: 29162642)
Genet Med. 2015 May;17(5):405-24. (PMID: 25741868)
Ophthalmology. 2016 Jun;123(6):1375-85. (PMID: 26976702)
Genet Med. 2019 Aug;21(8):1751-1760. (PMID: 30643219)
Arch Soc Esp Oftalmol (Engl Ed). 2021 Nov;96 Suppl 1:4-14. (PMID: 34836588)
Mol Cell Probes. 1997 Aug;11(4):287-92. (PMID: 9281415)
J Appl Genet. 2018 Aug;59(3):253-268. (PMID: 29680930)
Prog Retin Eye Res. 2012 Sep;31(5):351-76. (PMID: 22561546)
Bioessays. 2002 Nov;24(11):1041-51. (PMID: 12386935)
J Med Genet. 2022 May;59(5):428-437. (PMID: 33782094)
Eur J Hum Genet. 1999 Apr;7(3):274-86. (PMID: 10234503)
Exp Eye Res. 2017 Mar;156:10-21. (PMID: 27126352)
Hum Mutat. 2015 Feb;36(2):210-21. (PMID: 25382762)
Front Cell Neurosci. 2020 Aug 20;14:265. (PMID: 32973457)
Methods. 2005 Dec;37(4):331-40. (PMID: 16314262)
Stem Cell Res Ther. 2021 Nov 22;12(1):581. (PMID: 34809716)
Genome Med. 2022 Feb 28;14(1):23. (PMID: 35220969)
Int J Mol Sci. 2021 May 13;22(10):. (PMID: 34068052)
Cell Stem Cell. 2016 Jun 2;18(6):769-781. (PMID: 27151457)
Cell. 2019 Jan 24;176(3):535-548.e24. (PMID: 30661751)
Dev Biol. 2008 Jan 1;313(1):434-54. (PMID: 18062951)
Front Genet. 2021 Jul 01;12:689892. (PMID: 34276790)
Eur J Hum Genet. 2019 Mar;27(3):488-493. (PMID: 30315214)
Mol Vis. 2012;18:751-7. (PMID: 22509105)
Bioinformatics. 2005 May 1;21(9):1859-75. (PMID: 15728110)
Stem Cell Res. 2021 Mar;51:102184. (PMID: 33524672)
Genome Biol. 2015 Sep 30;16:204. (PMID: 26420219)
Cells. 2022 Nov 06;11(21):. (PMID: 36359912)
Methods Mol Biol. 2022;2434:245-255. (PMID: 35213022)
Nucleic Acids Res. 2009 May;37(9):e67. (PMID: 19339519)
Stem Cell Res. 2019 Dec;41:101611. (PMID: 31707209)
Transl Res. 2015 Dec;166(6):740-749.e1. (PMID: 26364624)
Invest Ophthalmol Vis Sci. 2009 Jun;50(6):2581-90. (PMID: 19218613)
J Appl Genet. 2018 May;59(2):151-159. (PMID: 29460221)
Prog Retin Eye Res. 2021 Jul;83:100918. (PMID: 33130253)
Sci Rep. 2016 Aug 24;6:31602. (PMID: 27554526)
Hum Mol Genet. 1993 Jul;2(7):915-20. (PMID: 8364574)
Eur J Hum Genet. 2012 Oct;20(10):1011-7. (PMID: 22692063)
BMC Genet. 2005 May 26;6:27. (PMID: 15918896)
معلومات مُعتمدة: PI17/00164 Instituto de Salud Carlos III; PI20/00851 Instituto de Salud Carlos III; FI18/00123 Instituto de Salud Carlos III; CP16/00116 Instituto de Salud Carlos III; 06/07/0036 Centre for Biomedical Network Research on Rare Diseases; PT20/00141 Instituto de Salud Carlos III; NA Fundación ONCE; PEJD-2018-PRE/BMD-9453 Comunidad de Madrid; PEJ-2020-AI/BMD-18610 Comunidad de Madrid; PEJ-2017-AI/BMD-7256 Comunidad de Madrid; University Chair UAM-IIS-FJD of Genomic Medicine Autonomous University of Madrid
فهرسة مساهمة: Keywords: MinION nanopore sequencing; PAX6; congenital aniridia; deep-intronic variants; long-read sequencing; minigene splicing assays; non-canonical splicing sites
المشرفين على المادة: 0 (PAX6 protein, human)
0 (PAX6 Transcription Factor)
0 (RNA Splice Sites)
تواريخ الأحداث: Date Created: 20230121 Date Completed: 20230127 Latest Revision: 20230308
رمز التحديث: 20240628
مُعرف محوري في PubMed: PMC9863980
DOI: 10.3390/ijms24021562
PMID: 36675087
قاعدة البيانات: MEDLINE
الوصف
تدمد:1422-0067
DOI:10.3390/ijms24021562