In silico and biological analyses of missense variants of the human biliary efflux transporter ABCC2: effects of novel rare missense variants
Charlotte Kölz
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany
University of Tuebingen, Tuebingen, Germany
Cluster of Excellence iFIT (EXC 2180) “Image-Guided and Functionally Instructed Tumor Therapies”, University of Tübingen, Tübingen, Germany
Contribution: Conceptualization (equal), Formal analysis (equal), Investigation (equal), Methodology (equal), Writing - original draft (equal), Writing - review & editing (equal)
Search for more papers by this authorFabienne Z. Gaugaz
Department of Pharmacy, Uppsala University, Uppsala, Sweden
Contribution: Formal analysis (equal), Funding acquisition (equal), Investigation (equal), Methodology (equal), Writing - review & editing (equal)
Search for more papers by this authorNiklas Handin
Department of Pharmacy, Uppsala University, Uppsala, Sweden
Contribution: Formal analysis (equal), Investigation (equal), Methodology (equal), Writing - review & editing (equal)
Search for more papers by this authorElke Schaeffeler
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany
University of Tuebingen, Tuebingen, Germany
Cluster of Excellence iFIT (EXC 2180) “Image-Guided and Functionally Instructed Tumor Therapies”, University of Tübingen, Tübingen, Germany
Contribution: Conceptualization (equal), Formal analysis (equal), Funding acquisition (equal), Investigation (equal), Methodology (equal), Writing - review & editing (equal)
Search for more papers by this authorRoman Tremmel
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany
University of Tuebingen, Tuebingen, Germany
Contribution: Formal analysis (equal), Investigation (equal), Writing - review & editing (equal)
Search for more papers by this authorStefan Winter
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany
University of Tuebingen, Tuebingen, Germany
Contribution: Formal analysis (equal), Investigation (equal), Writing - review & editing (equal)
Search for more papers by this authorKathrin Klein
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany
University of Tuebingen, Tuebingen, Germany
Contribution: Formal analysis (equal), Investigation (equal), Writing - review & editing (equal)
Search for more papers by this authorUlrich M. Zanger
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany
University of Tuebingen, Tuebingen, Germany
Contribution: Formal analysis (equal), Methodology (equal), Writing - review & editing (equal)
Search for more papers by this authorPer Artursson
Department of Pharmacy, Uppsala University, Uppsala, Sweden
Contribution: Formal analysis (equal), Investigation (equal), Writing - review & editing (equal)
Search for more papers by this authorCorresponding Author
Matthias Schwab
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany
University of Tuebingen, Tuebingen, Germany
Cluster of Excellence iFIT (EXC 2180) “Image-Guided and Functionally Instructed Tumor Therapies”, University of Tübingen, Tübingen, Germany
Department of Clinical Pharmacology, Pharmacy and Biochemistry, University of Tuebingen, Tuebingen, Germany
Correspondence
Anne T. Nies and Matthias Schwab, Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstrasse 112, D-70376 Stuttgart, Germany.
Email: [email protected] and
Contribution: Conceptualization (equal), Funding acquisition (equal), Supervision (equal), Writing - review & editing (equal)
Search for more papers by this authorCorresponding Author
Anne T. Nies
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany
University of Tuebingen, Tuebingen, Germany
Cluster of Excellence iFIT (EXC 2180) “Image-Guided and Functionally Instructed Tumor Therapies”, University of Tübingen, Tübingen, Germany
Correspondence
Anne T. Nies and Matthias Schwab, Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstrasse 112, D-70376 Stuttgart, Germany.
Email: [email protected] and
Contribution: Conceptualization (equal), Formal analysis (equal), Funding acquisition (equal), Investigation (equal), Methodology (equal), Writing - original draft (equal), Writing - review & editing (equal)
Search for more papers by this authorCharlotte Kölz
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany
University of Tuebingen, Tuebingen, Germany
Cluster of Excellence iFIT (EXC 2180) “Image-Guided and Functionally Instructed Tumor Therapies”, University of Tübingen, Tübingen, Germany
Contribution: Conceptualization (equal), Formal analysis (equal), Investigation (equal), Methodology (equal), Writing - original draft (equal), Writing - review & editing (equal)
Search for more papers by this authorFabienne Z. Gaugaz
Department of Pharmacy, Uppsala University, Uppsala, Sweden
Contribution: Formal analysis (equal), Funding acquisition (equal), Investigation (equal), Methodology (equal), Writing - review & editing (equal)
Search for more papers by this authorNiklas Handin
Department of Pharmacy, Uppsala University, Uppsala, Sweden
Contribution: Formal analysis (equal), Investigation (equal), Methodology (equal), Writing - review & editing (equal)
Search for more papers by this authorElke Schaeffeler
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany
University of Tuebingen, Tuebingen, Germany
Cluster of Excellence iFIT (EXC 2180) “Image-Guided and Functionally Instructed Tumor Therapies”, University of Tübingen, Tübingen, Germany
Contribution: Conceptualization (equal), Formal analysis (equal), Funding acquisition (equal), Investigation (equal), Methodology (equal), Writing - review & editing (equal)
Search for more papers by this authorRoman Tremmel
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany
University of Tuebingen, Tuebingen, Germany
Contribution: Formal analysis (equal), Investigation (equal), Writing - review & editing (equal)
Search for more papers by this authorStefan Winter
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany
University of Tuebingen, Tuebingen, Germany
Contribution: Formal analysis (equal), Investigation (equal), Writing - review & editing (equal)
Search for more papers by this authorKathrin Klein
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany
University of Tuebingen, Tuebingen, Germany
Contribution: Formal analysis (equal), Investigation (equal), Writing - review & editing (equal)
Search for more papers by this authorUlrich M. Zanger
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany
University of Tuebingen, Tuebingen, Germany
Contribution: Formal analysis (equal), Methodology (equal), Writing - review & editing (equal)
Search for more papers by this authorPer Artursson
Department of Pharmacy, Uppsala University, Uppsala, Sweden
Contribution: Formal analysis (equal), Investigation (equal), Writing - review & editing (equal)
Search for more papers by this authorCorresponding Author
Matthias Schwab
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany
University of Tuebingen, Tuebingen, Germany
Cluster of Excellence iFIT (EXC 2180) “Image-Guided and Functionally Instructed Tumor Therapies”, University of Tübingen, Tübingen, Germany
Department of Clinical Pharmacology, Pharmacy and Biochemistry, University of Tuebingen, Tuebingen, Germany
Correspondence
Anne T. Nies and Matthias Schwab, Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstrasse 112, D-70376 Stuttgart, Germany.
Email: [email protected] and
Contribution: Conceptualization (equal), Funding acquisition (equal), Supervision (equal), Writing - review & editing (equal)
Search for more papers by this authorCorresponding Author
Anne T. Nies
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany
University of Tuebingen, Tuebingen, Germany
Cluster of Excellence iFIT (EXC 2180) “Image-Guided and Functionally Instructed Tumor Therapies”, University of Tübingen, Tübingen, Germany
Correspondence
Anne T. Nies and Matthias Schwab, Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstrasse 112, D-70376 Stuttgart, Germany.
Email: [email protected] and
Contribution: Conceptualization (equal), Formal analysis (equal), Funding acquisition (equal), Investigation (equal), Methodology (equal), Writing - original draft (equal), Writing - review & editing (equal)
Search for more papers by this authorAbstract
Background and Purpose
The ATP-dependent biliary efflux transporter ABCC2, also known as multidrug resistance protein 2 (MRP2), is essential for the cellular disposition and detoxification of various xenobiotics including drugs as well as endogenous metabolites. Common functionally relevant ABCC2 genetic variants significantly alter drug responses and contribute to side effects. The aim of this study was to determine functional consequences of rare variants identified in subjects with European ancestry using in silico tools and in vitro analyses.
Experimental Approach
Targeted next-generation sequencing of the ABCC2 gene was used to identify novel variants in European subjects (n = 143). Twenty-six in silico tools were used to predict functional consequences. For biological validation, transport assays were carried out with membrane vesicles prepared from cell lines overexpressing the newly identified ABCC2 variants and estradiol β-glucuronide and carboxydichlorofluorescein as the substrates.
Key Results
Three novel rare ABCC2 missense variants were identified (W227R, K402T, V489F). Twenty-five in silico tools predicted W227R as damaging and one as potentially damaging. Prediction of functional consequences was not possible for K402T and V489F and for the common linked variants V1188E/C1515Y. Characterisation in vitro showed increased function of W227R, V489F and V1188E/C1515Y for both substrates, whereas K402T function was only increased for carboxydichlorofluorescein.
Conclusion and Implications
In silico tools were unable to accurately predict the substrate-dependent increase in function of ABCC2 missense variants. In vitro biological studies are required to accurately determine functional activity to avoid misleading consequences for drug therapy.
CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.
Open Research
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.
Supporting Information
| Filename | Description |
|---|---|
| bph16508-sup-0001-Supporting_Information.pdfPDF document, 418.1 KB |
Table S1. In silico tools and criteria for prediction scores. Table S2. Surrogate peptides and their mass transitions. Table S3. Minor allele frequencies (MAF) and other annotations of the ABCC2 exonic variants identified in the human liver cohort in comparison to the data for non-Finnish Europeans from the Genome Aggregation Database gnomAD. |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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