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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Frontiers Media S.A.
2020-03-01
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| Series: | Frontiers in Genetics |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fgene.2020.00169/full |
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| author | Linda Gailite Alberto Valenzuela-Palomo Lara Sanoguera-Miralles Dmitrijs Rots Madara Kreile Eladio A. Velasco |
| author_facet | Linda Gailite Alberto Valenzuela-Palomo Lara Sanoguera-Miralles Dmitrijs Rots Madara Kreile Eladio A. Velasco |
| author_sort | Linda Gailite |
| collection | DOAJ |
| 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. |
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| issn | 1664-8021 |
| language | English |
| last_indexed | 2024-12-10T21:21:48Z |
| publishDate | 2020-03-01 |
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| series | Frontiers in Genetics |
| spelling | doaj.art-1c8b08301f3148cc96b7f111e1d9ec0f2022-12-22T01:33:06ZengFrontiers Media S.A.Frontiers in Genetics1664-80212020-03-011110.3389/fgene.2020.00169512116UGT1A1 Variants c.864+5G>T and c.996+2_996+5del of a Crigler-Najjar Patient Induce Aberrant Splicing in Minigene AssaysLinda Gailite0Alberto Valenzuela-Palomo1Lara Sanoguera-Miralles2Dmitrijs Rots3Madara Kreile4Eladio A. Velasco5Scientific Laboratory of Molecular Genetics, Riga Stradins University, Riga, LatviaSplicing and Genetic Susceptibility to Cancer, Instituto de Biología y Genética Molecular (CSIC-UVa), Valladolid, SpainSplicing and Genetic Susceptibility to Cancer, Instituto de Biología y Genética Molecular (CSIC-UVa), Valladolid, SpainScientific Laboratory of Molecular Genetics, Riga Stradins University, Riga, LatviaScientific Laboratory of Molecular Genetics, Riga Stradins University, Riga, LatviaSplicing and Genetic Susceptibility to Cancer, Instituto de Biología y Genética Molecular (CSIC-UVa), Valladolid, SpainA 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.https://www.frontiersin.org/article/10.3389/fgene.2020.00169/fullUGT1A1unconjugated hyperbilirubinemiaCrigler-Najjaraberrant splicingminigenesplice site |
| spellingShingle | Linda Gailite Alberto Valenzuela-Palomo Lara Sanoguera-Miralles Dmitrijs Rots Madara Kreile Eladio A. Velasco UGT1A1 Variants c.864+5G>T and c.996+2_996+5del of a Crigler-Najjar Patient Induce Aberrant Splicing in Minigene Assays Frontiers in Genetics UGT1A1 unconjugated hyperbilirubinemia Crigler-Najjar aberrant splicing minigene splice site |
| title | 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_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_sort | ugt1a1 variants c 864 5g t and c 996 2 996 5del of a crigler najjar patient induce aberrant splicing in minigene assays |
| topic | UGT1A1 unconjugated hyperbilirubinemia Crigler-Najjar aberrant splicing minigene splice site |
| url | https://www.frontiersin.org/article/10.3389/fgene.2020.00169/full |
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