Effects of a Common Eight Base Pairs Duplication at the Exon 7-Intron 7 Junction on Splicing, Expression, and Function of OCT1
Organic cation transporter 1 (OCT1, SLC22A1) is localized in the sinusoidal membrane of human hepatocytes and mediates hepatic uptake of weakly basic or cationic drugs and endogenous compounds. Common amino acid substitutions in OCT1 were associated with altered pharmacokinetics and efficacy of drug...
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Frontiers Media S.A.
2021-05-01
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| Series: | Frontiers in Pharmacology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fphar.2021.661480/full |
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| author | Sarah Römer Marleen J. Meyer Kathrin Klein Kathrin Klein Lennart V. Schneider Johannes Matthaei Ana Tzvetkova Ana Tzvetkova Joanna Łapczuk-Romańska Jochen Gaedcke Marek Droździk Jürgen Brockmöller Anne T. Nies Anne T. Nies Anne T. Nies Mladen V. Tzvetkov |
| author_facet | Sarah Römer Marleen J. Meyer Kathrin Klein Kathrin Klein Lennart V. Schneider Johannes Matthaei Ana Tzvetkova Ana Tzvetkova Joanna Łapczuk-Romańska Jochen Gaedcke Marek Droździk Jürgen Brockmöller Anne T. Nies Anne T. Nies Anne T. Nies Mladen V. Tzvetkov |
| author_sort | Sarah Römer |
| collection | DOAJ |
| description | Organic cation transporter 1 (OCT1, SLC22A1) is localized in the sinusoidal membrane of human hepatocytes and mediates hepatic uptake of weakly basic or cationic drugs and endogenous compounds. Common amino acid substitutions in OCT1 were associated with altered pharmacokinetics and efficacy of drugs like sumatriptan and fenoterol. Recently, the common splice variant rs35854239 has also been suggested to affect OCT1 function. rs35854239 represents an 8 bp duplication of the donor splice site at the exon 7-intron 7 junction. Here we quantified the extent to which this duplication affects OCT1 splicing and, as a consequence, the expression and the function of OCT1. We used pyrosequencing and deep RNA-sequencing to quantify the effect of rs35854239 on splicing after minigene expression of this variant in HepG2 and Huh7 cells and directly in human liver samples. Further, we analyzed the effects of rs35854239 on OCT1 mRNA expression in total, localization and activity of the resulting OCT1 protein, and on the pharmacokinetics of sumatriptan and fenoterol. The 8 bp duplication caused alternative splicing in 38% (deep RNA-sequencing) to 52% (pyrosequencing) of the minigene transcripts when analyzed in HepG2 and Huh7 cells. The alternatively spliced transcript encodes for a truncated protein that after transient transfection in HEK293 cells was not localized in the plasma membrane and was not able to transport the OCT1 model substrate ASP+. In human liver, however, the alternatively spliced OCT1 transcript was detectable only at very low levels (0.3% in heterozygous and 0.6% in homozygous carriers of the 8 bp duplication, deep RNA-sequencing). The 8 bp duplication was associated with a significant reduction of OCT1 expression in the human liver, but explained only 9% of the general variability in OCT1 expression and was not associated with significant changes in the pharmacokinetics of sumatriptan and fenoterol. Therefore, the rs35854239 variant only partially changes splicing, causing moderate changes in OCT1 expression and may be of only limited therapeutic relevance. |
| first_indexed | 2024-12-17T07:37:03Z |
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| spelling | doaj.art-830b00b369d741069648215b61af6c252022-12-21T21:58:17ZengFrontiers Media S.A.Frontiers in Pharmacology1663-98122021-05-011210.3389/fphar.2021.661480661480Effects of a Common Eight Base Pairs Duplication at the Exon 7-Intron 7 Junction on Splicing, Expression, and Function of OCT1Sarah Römer0Marleen J. Meyer1Kathrin Klein2Kathrin Klein3Lennart V. Schneider4Johannes Matthaei5Ana Tzvetkova6Ana Tzvetkova7Joanna Łapczuk-Romańska8Jochen Gaedcke9Marek Droździk10Jürgen Brockmöller11Anne T. Nies12Anne T. Nies13Anne T. Nies14Mladen V. Tzvetkov15Institute of Pharmacology, Center of Drug Absorption and Transport (C_DAT), University Medicine Greifswald, Greifswald, GermanyInstitute of Pharmacology, Center of Drug Absorption and Transport (C_DAT), University Medicine Greifswald, Greifswald, GermanyDr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, GermanyUniversity of Tuebingen, Tuebingen, GermanyInstitute of Clinical Pharmacology, University Medical Center Göttingen, Göttingen, GermanyInstitute of Clinical Pharmacology, University Medical Center Göttingen, Göttingen, GermanyInstitute of Bioinformatics, University Medicine Greifswald, Greifswald, GermanyHuman Molecular Genetics Group, Department of Functional Genomics, Interfaculty Institute of Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, GermanyDepartment of Experimental and Clinical Pharmacology, Pomeranian Medical University, Szczecin, PolandDepartment of General, Visceral and Pediatric Surgery, University Medical Center Göttingen, Göttingen, GermanyDepartment of Experimental and Clinical Pharmacology, Pomeranian Medical University, Szczecin, PolandInstitute of Clinical Pharmacology, University Medical Center Göttingen, Göttingen, GermanyDr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, GermanyUniversity of Tuebingen, Tuebingen, GermanyCluster of Excellence iFIT (EXC2180) “Image-Guided and Functionally Instructed Tumor Therapies”, University of Tuebingen, Tuebingen, GermanyInstitute of Pharmacology, Center of Drug Absorption and Transport (C_DAT), University Medicine Greifswald, Greifswald, GermanyOrganic cation transporter 1 (OCT1, SLC22A1) is localized in the sinusoidal membrane of human hepatocytes and mediates hepatic uptake of weakly basic or cationic drugs and endogenous compounds. Common amino acid substitutions in OCT1 were associated with altered pharmacokinetics and efficacy of drugs like sumatriptan and fenoterol. Recently, the common splice variant rs35854239 has also been suggested to affect OCT1 function. rs35854239 represents an 8 bp duplication of the donor splice site at the exon 7-intron 7 junction. Here we quantified the extent to which this duplication affects OCT1 splicing and, as a consequence, the expression and the function of OCT1. We used pyrosequencing and deep RNA-sequencing to quantify the effect of rs35854239 on splicing after minigene expression of this variant in HepG2 and Huh7 cells and directly in human liver samples. Further, we analyzed the effects of rs35854239 on OCT1 mRNA expression in total, localization and activity of the resulting OCT1 protein, and on the pharmacokinetics of sumatriptan and fenoterol. The 8 bp duplication caused alternative splicing in 38% (deep RNA-sequencing) to 52% (pyrosequencing) of the minigene transcripts when analyzed in HepG2 and Huh7 cells. The alternatively spliced transcript encodes for a truncated protein that after transient transfection in HEK293 cells was not localized in the plasma membrane and was not able to transport the OCT1 model substrate ASP+. In human liver, however, the alternatively spliced OCT1 transcript was detectable only at very low levels (0.3% in heterozygous and 0.6% in homozygous carriers of the 8 bp duplication, deep RNA-sequencing). The 8 bp duplication was associated with a significant reduction of OCT1 expression in the human liver, but explained only 9% of the general variability in OCT1 expression and was not associated with significant changes in the pharmacokinetics of sumatriptan and fenoterol. Therefore, the rs35854239 variant only partially changes splicing, causing moderate changes in OCT1 expression and may be of only limited therapeutic relevance.https://www.frontiersin.org/articles/10.3389/fphar.2021.661480/fullins/del variantorganic cation transporter 1SLC22A1minigeneallelic expression imbalance (AEI)fenoterol |
| spellingShingle | Sarah Römer Marleen J. Meyer Kathrin Klein Kathrin Klein Lennart V. Schneider Johannes Matthaei Ana Tzvetkova Ana Tzvetkova Joanna Łapczuk-Romańska Jochen Gaedcke Marek Droździk Jürgen Brockmöller Anne T. Nies Anne T. Nies Anne T. Nies Mladen V. Tzvetkov Effects of a Common Eight Base Pairs Duplication at the Exon 7-Intron 7 Junction on Splicing, Expression, and Function of OCT1 Frontiers in Pharmacology ins/del variant organic cation transporter 1 SLC22A1 minigene allelic expression imbalance (AEI) fenoterol |
| title | Effects of a Common Eight Base Pairs Duplication at the Exon 7-Intron 7 Junction on Splicing, Expression, and Function of OCT1 |
| title_full | Effects of a Common Eight Base Pairs Duplication at the Exon 7-Intron 7 Junction on Splicing, Expression, and Function of OCT1 |
| title_fullStr | Effects of a Common Eight Base Pairs Duplication at the Exon 7-Intron 7 Junction on Splicing, Expression, and Function of OCT1 |
| title_full_unstemmed | Effects of a Common Eight Base Pairs Duplication at the Exon 7-Intron 7 Junction on Splicing, Expression, and Function of OCT1 |
| title_short | Effects of a Common Eight Base Pairs Duplication at the Exon 7-Intron 7 Junction on Splicing, Expression, and Function of OCT1 |
| title_sort | effects of a common eight base pairs duplication at the exon 7 intron 7 junction on splicing expression and function of oct1 |
| topic | ins/del variant organic cation transporter 1 SLC22A1 minigene allelic expression imbalance (AEI) fenoterol |
| url | https://www.frontiersin.org/articles/10.3389/fphar.2021.661480/full |
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