UGT1A1 variants c.864+5G>T and c.996+2_996+5del of a Crigler-Najjar patient induce aberrant splicing in minigene assays

A large fraction of DNA variants impairs pre-mRNA splicing in human hereditary disorders. Crigler-Najjar syndrome (CNS) is characterized by a severe unconjugated hyperbilirubinemia caused by variants in the UGT1A1 gene. We previously reported one CNS-type II patient with two splice-site variants in...

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Autores: Gailite, Linda, Valenzuela-Palomo, Alberto, Sanoguera-Miralles, Lara, Rots, Dmitrijs, Kreile, Madara, Velasco, Eladio
Tipo de documento: artigo
Estado:Versão publicada
Data de publicação:2020
País:España
Recursos:Consejo Superior de Investigaciones Científicas (CSIC)
Repositório:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/223205
Acesso em linha:http://hdl.handle.net/10261/223205
Access Level:Acceso aberto
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spelling UGT1A1 variants c.864+5G>T and c.996+2_996+5del of a Crigler-Najjar patient induce aberrant splicing in minigene assaysGailite, LindaValenzuela-Palomo, AlbertoSanoguera-Miralles, LaraRots, DmitrijsKreile, MadaraVelasco, EladioA large fraction of DNA variants impairs pre-mRNA splicing in human hereditary disorders. Crigler-Najjar syndrome (CNS) is characterized by a severe unconjugated hyperbilirubinemia caused by variants in the UGT1A1 gene. We previously reported one CNS-type II patient with two splice-site variants in trans (c.864+5G>T and c.996+2_996+5del). According to MaxEntScan, both disrupt their corresponding donor sites (c.864+5G>T: 6.99 → 2.28; c.996+2_996+5del: 5.96 → −11.02), so they were selected for subsequent functional tests. Given the unavailability of patient RNA, we constructed an UGT1A1 splicing-reporter minigene with exons 1–4 to characterize the underlying splicing anomaly. The variant c.996+2_996+5del generated two aberrant transcripts, Δ(E2) (exon 2 skipping/64%) and ▼(E2q135) (intron retention of 135-nt/36%), which lead to the loss of 18 conserved amino-acids and the gain of 45 new ones of a critical functional domain, respectively. The c.864+5G>T variant mainly produced the aberrant transcript Δ(E1q141) (141-nt deletion/70.4%) and the full-length isoform (29.6%). Δ(E1q141) would provoke the loss of 47 amino-acids of the N-terminal domain that encodes for substrate specificity. Thus, the three anomalous transcripts are likely to inactivate UGT1A1. Moreover, this patient is also homozygous for the promoter variant A(TA)7TAA that decreases UGT1A1 expression by 70%, so the full-length transcript produced by c.864+5G>T would be even more reduced (<9%), thus supporting the diagnosis of CNS-type II. Therefore, minigenes represent valuable tools for the functional and clinical classifications of genetic variants.The Scientific Laboratory of Molecular Genetics was funded by internal grants from the Riga Stradins University. EV was supported by grants from the Spanish Ministry of Science, Innovation and Universities, Plan Nacional de I + D + I 2013-2016, ISCIII (PI17/00227) co-funded by FEDER from Regional Development European Funds (European Union), and grant CSI242P18 (actuación cofinanciada P.O. FEDER 2014-2020 de Castilla y León) from the Consejería de Educación, Junta de Castilla y León. AV-P was supported by a predoctoral fellowship from the Consejería de Educación, Junta de Castilla y León (2018–2022). LS-M was supported by a predoctoral fellowship from the Spanish Association Against Cancer (AECC), Junta Provincial de Valladolid (2019–2023).Peer reviewedFrontiers MediaInstituto de Salud Carlos IIIAgencia Estatal de Investigación (España)Ministerio de Ciencia, Innovación y Universidades (España)Junta de Castilla y LeónEuropean CommissionAsociación Española Contra el CáncerConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]202020202020info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/223205reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Ingléshttps://doi.org/10.3389/fgene.2020.00169Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/2232052026-05-22T06:33:51Z
dc.title.none.fl_str_mv UGT1A1 variants c.864+5G>T and c.996+2_996+5del of a Crigler-Najjar patient induce aberrant splicing in minigene assays
title UGT1A1 variants c.864+5G>T and c.996+2_996+5del of a Crigler-Najjar patient induce aberrant splicing in minigene assays
spellingShingle UGT1A1 variants c.864+5G>T and c.996+2_996+5del of a Crigler-Najjar patient induce aberrant splicing in minigene assays
Gailite, Linda
title_short UGT1A1 variants c.864+5G>T and c.996+2_996+5del of a Crigler-Najjar patient induce aberrant splicing in minigene assays
title_full UGT1A1 variants c.864+5G>T and c.996+2_996+5del of a Crigler-Najjar patient induce aberrant splicing in minigene assays
title_fullStr UGT1A1 variants c.864+5G>T and c.996+2_996+5del of a Crigler-Najjar patient induce aberrant splicing in minigene assays
title_full_unstemmed UGT1A1 variants c.864+5G>T and c.996+2_996+5del of a Crigler-Najjar patient induce aberrant splicing in minigene assays
title_sort UGT1A1 variants c.864+5G>T and c.996+2_996+5del of a Crigler-Najjar patient induce aberrant splicing in minigene assays
dc.creator.none.fl_str_mv Gailite, Linda
Valenzuela-Palomo, Alberto
Sanoguera-Miralles, Lara
Rots, Dmitrijs
Kreile, Madara
Velasco, Eladio
author Gailite, Linda
author_facet Gailite, Linda
Valenzuela-Palomo, Alberto
Sanoguera-Miralles, Lara
Rots, Dmitrijs
Kreile, Madara
Velasco, Eladio
author_role author
author2 Valenzuela-Palomo, Alberto
Sanoguera-Miralles, Lara
Rots, Dmitrijs
Kreile, Madara
Velasco, Eladio
author2_role author
author
author
author
author
dc.contributor.none.fl_str_mv Instituto de Salud Carlos III
Agencia Estatal de Investigación (España)
Ministerio de Ciencia, Innovación y Universidades (España)
Junta de Castilla y León
European Commission
Asociación Española Contra el Cáncer
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
description A large fraction of DNA variants impairs pre-mRNA splicing in human hereditary disorders. Crigler-Najjar syndrome (CNS) is characterized by a severe unconjugated hyperbilirubinemia caused by variants in the UGT1A1 gene. We previously reported one CNS-type II patient with two splice-site variants in trans (c.864+5G>T and c.996+2_996+5del). According to MaxEntScan, both disrupt their corresponding donor sites (c.864+5G>T: 6.99 → 2.28; c.996+2_996+5del: 5.96 → −11.02), so they were selected for subsequent functional tests. Given the unavailability of patient RNA, we constructed an UGT1A1 splicing-reporter minigene with exons 1–4 to characterize the underlying splicing anomaly. The variant c.996+2_996+5del generated two aberrant transcripts, Δ(E2) (exon 2 skipping/64%) and ▼(E2q135) (intron retention of 135-nt/36%), which lead to the loss of 18 conserved amino-acids and the gain of 45 new ones of a critical functional domain, respectively. The c.864+5G>T variant mainly produced the aberrant transcript Δ(E1q141) (141-nt deletion/70.4%) and the full-length isoform (29.6%). Δ(E1q141) would provoke the loss of 47 amino-acids of the N-terminal domain that encodes for substrate specificity. Thus, the three anomalous transcripts are likely to inactivate UGT1A1. Moreover, this patient is also homozygous for the promoter variant A(TA)7TAA that decreases UGT1A1 expression by 70%, so the full-length transcript produced by c.864+5G>T would be even more reduced (<9%), thus supporting the diagnosis of CNS-type II. Therefore, minigenes represent valuable tools for the functional and clinical classifications of genetic variants.
publishDate 2020
dc.date.none.fl_str_mv 2020
2020
2020
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/223205
url http://hdl.handle.net/10261/223205
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv https://doi.org/10.3389/fgene.2020.00169
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Frontiers Media
publisher.none.fl_str_mv Frontiers Media
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
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repository.mail.fl_str_mv
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