SLCO1B1 : Application and Limitations of Deep Mutational Scanning for Genomic Missense Variant Function
( ) is an important transmembrane hepatic uptake transporter. Genetic variants in the gene have been associated with altered protein folding, resulting in protein degradation and decreased transporter activity. Next-generation sequencing (NGS) of pharmacogenes is being applied increasingly to associ...
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| Published in: | Drug metabolism and disposition Vol. 49; no. 5; p. 395 |
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| Main Authors: | , , , , , , |
| Format: | Journal Article |
| Language: | English |
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01.05.2021
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| ISSN: | 1521-009X, 1521-009X |
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| Abstract | (
) is an important transmembrane hepatic uptake transporter. Genetic variants in the
gene have been associated with altered protein folding, resulting in protein degradation and decreased transporter activity. Next-generation sequencing (NGS) of pharmacogenes is being applied increasingly to associate variation in drug response with genetic sequence variants. However, it is difficult to link variants of unknown significance with functional phenotypes using "one-at-a-time" functional systems. Deep mutational scanning (DMS) using a "landing pad cell-based system" is a high-throughput technique designed to analyze hundreds of gene open reading frame (ORF) missense variants in a parallel and scalable fashion. We have applied DMS to analyze 137 missense variants in the
ORF obtained from the Exome Aggregation Consortium project. ORFs containing these variants were fused to green fluorescent protein and were integrated into "landing pad" cells. Florescence-activated cell sorting was performed to separate the cells into four groups based on fluorescence readout indicating protein expression at the single cell level. NGS was then performed and
variant frequencies were used to determine protein abundance. We found that six variants not previously characterized functionally displayed less than 25% and another 12 displayed approximately 50% of wild-type protein expression. These results were then functionally validated by transporter studies. Severely damaging variants identified by DMS may have clinical relevance for
dependent drug transport, but we need to exercise caution since the relatively small number of severely damaging variants identified raise questions with regard to the application of DMS to intrinsic membrane proteins such as organic anion transporter protein 1B1. SIGNIFICANCE STATEMENT: The functional implications of a large numbers of open reading frame (ORF) "variants of unknown significance" (VUS) in transporter genes have not been characterized. This study applied deep mutational scanning to determine the functional effects of VUS that have been observed in the ORF of
(
). Several severely damaging variants were identified, studied, and validated. These observations have implications for both the application of deep mutational scanning to intrinsic membrane proteins and for the clinical effect of drugs and endogenous compounds transported by
. |
|---|---|
| AbstractList | (
) is an important transmembrane hepatic uptake transporter. Genetic variants in the
gene have been associated with altered protein folding, resulting in protein degradation and decreased transporter activity. Next-generation sequencing (NGS) of pharmacogenes is being applied increasingly to associate variation in drug response with genetic sequence variants. However, it is difficult to link variants of unknown significance with functional phenotypes using "one-at-a-time" functional systems. Deep mutational scanning (DMS) using a "landing pad cell-based system" is a high-throughput technique designed to analyze hundreds of gene open reading frame (ORF) missense variants in a parallel and scalable fashion. We have applied DMS to analyze 137 missense variants in the
ORF obtained from the Exome Aggregation Consortium project. ORFs containing these variants were fused to green fluorescent protein and were integrated into "landing pad" cells. Florescence-activated cell sorting was performed to separate the cells into four groups based on fluorescence readout indicating protein expression at the single cell level. NGS was then performed and
variant frequencies were used to determine protein abundance. We found that six variants not previously characterized functionally displayed less than 25% and another 12 displayed approximately 50% of wild-type protein expression. These results were then functionally validated by transporter studies. Severely damaging variants identified by DMS may have clinical relevance for
dependent drug transport, but we need to exercise caution since the relatively small number of severely damaging variants identified raise questions with regard to the application of DMS to intrinsic membrane proteins such as organic anion transporter protein 1B1. SIGNIFICANCE STATEMENT: The functional implications of a large numbers of open reading frame (ORF) "variants of unknown significance" (VUS) in transporter genes have not been characterized. This study applied deep mutational scanning to determine the functional effects of VUS that have been observed in the ORF of
(
). Several severely damaging variants were identified, studied, and validated. These observations have implications for both the application of deep mutational scanning to intrinsic membrane proteins and for the clinical effect of drugs and endogenous compounds transported by
. SLCO1B1 (solute carrier organic anion transporter family member 1B1) is an important transmembrane hepatic uptake transporter. Genetic variants in the SLCO1B1 gene have been associated with altered protein folding, resulting in protein degradation and decreased transporter activity. Next-generation sequencing (NGS) of pharmacogenes is being applied increasingly to associate variation in drug response with genetic sequence variants. However, it is difficult to link variants of unknown significance with functional phenotypes using "one-at-a-time" functional systems. Deep mutational scanning (DMS) using a "landing pad cell-based system" is a high-throughput technique designed to analyze hundreds of gene open reading frame (ORF) missense variants in a parallel and scalable fashion. We have applied DMS to analyze 137 missense variants in the SLCO1B1 ORF obtained from the Exome Aggregation Consortium project. ORFs containing these variants were fused to green fluorescent protein and were integrated into "landing pad" cells. Florescence-activated cell sorting was performed to separate the cells into four groups based on fluorescence readout indicating protein expression at the single cell level. NGS was then performed and SLCO1B1 variant frequencies were used to determine protein abundance. We found that six variants not previously characterized functionally displayed less than 25% and another 12 displayed approximately 50% of wild-type protein expression. These results were then functionally validated by transporter studies. Severely damaging variants identified by DMS may have clinical relevance for SLCO1B1-dependent drug transport, but we need to exercise caution since the relatively small number of severely damaging variants identified raise questions with regard to the application of DMS to intrinsic membrane proteins such as organic anion transporter protein 1B1. SIGNIFICANCE STATEMENT: The functional implications of a large numbers of open reading frame (ORF) "variants of unknown significance" (VUS) in transporter genes have not been characterized. This study applied deep mutational scanning to determine the functional effects of VUS that have been observed in the ORF of SLCO1B1(s olute carrier organic anion transporter family member 1B1). Several severely damaging variants were identified, studied, and validated. These observations have implications for both the application of deep mutational scanning to intrinsic membrane proteins and for the clinical effect of drugs and endogenous compounds transported by SLCO1B1.SLCO1B1 (solute carrier organic anion transporter family member 1B1) is an important transmembrane hepatic uptake transporter. Genetic variants in the SLCO1B1 gene have been associated with altered protein folding, resulting in protein degradation and decreased transporter activity. Next-generation sequencing (NGS) of pharmacogenes is being applied increasingly to associate variation in drug response with genetic sequence variants. However, it is difficult to link variants of unknown significance with functional phenotypes using "one-at-a-time" functional systems. Deep mutational scanning (DMS) using a "landing pad cell-based system" is a high-throughput technique designed to analyze hundreds of gene open reading frame (ORF) missense variants in a parallel and scalable fashion. We have applied DMS to analyze 137 missense variants in the SLCO1B1 ORF obtained from the Exome Aggregation Consortium project. ORFs containing these variants were fused to green fluorescent protein and were integrated into "landing pad" cells. Florescence-activated cell sorting was performed to separate the cells into four groups based on fluorescence readout indicating protein expression at the single cell level. NGS was then performed and SLCO1B1 variant frequencies were used to determine protein abundance. We found that six variants not previously characterized functionally displayed less than 25% and another 12 displayed approximately 50% of wild-type protein expression. These results were then functionally validated by transporter studies. Severely damaging variants identified by DMS may have clinical relevance for SLCO1B1-dependent drug transport, but we need to exercise caution since the relatively small number of severely damaging variants identified raise questions with regard to the application of DMS to intrinsic membrane proteins such as organic anion transporter protein 1B1. SIGNIFICANCE STATEMENT: The functional implications of a large numbers of open reading frame (ORF) "variants of unknown significance" (VUS) in transporter genes have not been characterized. This study applied deep mutational scanning to determine the functional effects of VUS that have been observed in the ORF of SLCO1B1(s olute carrier organic anion transporter family member 1B1). Several severely damaging variants were identified, studied, and validated. These observations have implications for both the application of deep mutational scanning to intrinsic membrane proteins and for the clinical effect of drugs and endogenous compounds transported by SLCO1B1. |
| Author | Zhang, Lingxin Kalari, Krishna R Weinshilboum, Richard M Moon, Irene Sarangi, Vivekananda Wang, Liewei Ho, Ming-Fen |
| Author_xml | – sequence: 1 givenname: Lingxin surname: Zhang fullname: Zhang, Lingxin organization: Division of Clinical Pharmacology, Department of Molecular Pharmacology and Experimental Therapeutics (L.Z., M.-F.H., I.M., L.W., R.M.W.), Division of Biomedical Statistics and Informatics, Department of Health Sciences Research (V.S., K.R.K.), and Mayo Clinic Center for Individualized Medicine (L.W., R.M.W.), Mayo Clinic, Rochester, Minnesota – sequence: 2 givenname: Vivekananda surname: Sarangi fullname: Sarangi, Vivekananda organization: Division of Clinical Pharmacology, Department of Molecular Pharmacology and Experimental Therapeutics (L.Z., M.-F.H., I.M., L.W., R.M.W.), Division of Biomedical Statistics and Informatics, Department of Health Sciences Research (V.S., K.R.K.), and Mayo Clinic Center for Individualized Medicine (L.W., R.M.W.), Mayo Clinic, Rochester, Minnesota – sequence: 3 givenname: Ming-Fen surname: Ho fullname: Ho, Ming-Fen organization: Division of Clinical Pharmacology, Department of Molecular Pharmacology and Experimental Therapeutics (L.Z., M.-F.H., I.M., L.W., R.M.W.), Division of Biomedical Statistics and Informatics, Department of Health Sciences Research (V.S., K.R.K.), and Mayo Clinic Center for Individualized Medicine (L.W., R.M.W.), Mayo Clinic, Rochester, Minnesota – sequence: 4 givenname: Irene surname: Moon fullname: Moon, Irene organization: Division of Clinical Pharmacology, Department of Molecular Pharmacology and Experimental Therapeutics (L.Z., M.-F.H., I.M., L.W., R.M.W.), Division of Biomedical Statistics and Informatics, Department of Health Sciences Research (V.S., K.R.K.), and Mayo Clinic Center for Individualized Medicine (L.W., R.M.W.), Mayo Clinic, Rochester, Minnesota – sequence: 5 givenname: Krishna R surname: Kalari fullname: Kalari, Krishna R organization: Division of Clinical Pharmacology, Department of Molecular Pharmacology and Experimental Therapeutics (L.Z., M.-F.H., I.M., L.W., R.M.W.), Division of Biomedical Statistics and Informatics, Department of Health Sciences Research (V.S., K.R.K.), and Mayo Clinic Center for Individualized Medicine (L.W., R.M.W.), Mayo Clinic, Rochester, Minnesota – sequence: 6 givenname: Liewei surname: Wang fullname: Wang, Liewei email: weinshilboum.richard@mayo.edu, Wang.Liewei@mayo.edu organization: Division of Clinical Pharmacology, Department of Molecular Pharmacology and Experimental Therapeutics (L.Z., M.-F.H., I.M., L.W., R.M.W.), Division of Biomedical Statistics and Informatics, Department of Health Sciences Research (V.S., K.R.K.), and Mayo Clinic Center for Individualized Medicine (L.W., R.M.W.), Mayo Clinic, Rochester, Minnesota weinshilboum.richard@mayo.edu Wang.Liewei@mayo.edu – sequence: 7 givenname: Richard M surname: Weinshilboum fullname: Weinshilboum, Richard M email: weinshilboum.richard@mayo.edu, Wang.Liewei@mayo.edu organization: Division of Clinical Pharmacology, Department of Molecular Pharmacology and Experimental Therapeutics (L.Z., M.-F.H., I.M., L.W., R.M.W.), Division of Biomedical Statistics and Informatics, Department of Health Sciences Research (V.S., K.R.K.), and Mayo Clinic Center for Individualized Medicine (L.W., R.M.W.), Mayo Clinic, Rochester, Minnesota weinshilboum.richard@mayo.edu Wang.Liewei@mayo.edu |
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) is an important transmembrane hepatic uptake transporter. Genetic variants in the
gene have been associated with altered protein folding, resulting in... SLCO1B1 (solute carrier organic anion transporter family member 1B1) is an important transmembrane hepatic uptake transporter. Genetic variants in the SLCO1B1... |
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| SubjectTerms | Genetic Variation - genetics Genomics - methods HEK293 Cells Humans Liver-Specific Organic Anion Transporter 1 - genetics Mutation, Missense - genetics |
| Title | SLCO1B1 : Application and Limitations of Deep Mutational Scanning for Genomic Missense Variant Function |
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