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.
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
Drug Metab Dispos. 2021 May;49(5):395-404. doi: 10.1124/dmd.120.000264. Epub 2021 Mar 3.
() 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 .
（）是一种重要的跨膜肝摄取转运体。基因中的 基因变异与蛋白质折叠改变有关，导致蛋白质降解和转运体活性降低。下一代测序（NGS）正在越来越多地应用于将药物反应的变异与遗传序列变异相关联。然而，使用“逐个”功能系统很难将具有未知意义的变体与功能表型联系起来。使用“着陆垫细胞为基础的系统”的深度突变扫描（DMS）是一种高通量技术，旨在以平行和可扩展的方式分析数百个基因开放阅读框（ORF）错义变体。我们应用 DMS 分析了从外显子聚集协会项目获得的 基因的 137 个错义变体。包含这些变体的 ORFs 与绿色荧光蛋白融合，并整合到“着陆垫”细胞中。荧光激活细胞分选（FACS）用于根据荧光读数将细胞分为四个组，该读数指示单细胞水平的蛋白质表达。然后进行 NGS，并使用变体频率确定蛋白质丰度。我们发现，六个以前未被功能表征的变体显示出低于 25%的表达，另外 12 个变体显示出约 50%的野生型蛋白表达。这些结果随后通过转运蛋白研究进行了功能验证。通过 DMS 鉴定的严重破坏性变体可能对依赖药物的转运具有临床相关性，但我们需要谨慎，因为 DMS 应用于有机阴离子转运蛋白 1B1 等内在膜蛋白时，所鉴定的严重破坏性变体数量相对较少，这引发了一些问题。意义声明：转运体基因中大量“未知意义的变异体（VUS）”的功能意义尚未得到描述。本研究应用深度突变扫描来确定在 基因的 ORF 中观察到的 VUS 的功能影响。鉴定出了几个严重破坏性变体，并进行了研究和验证。这些观察结果对深度突变扫描应用于内在膜蛋白以及药物和内源性化合物的临床效果都有影响。
