SLCO1B1: Application and Limitations of Deep Mutational Scanning for Genomic Missense Variant Function.
SLCO1B1: Application and Limitations of Deep Mutational Scanning for Genomic Missense Variant Function.
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SLCO1B1：对基因组错义变异功能的深突变扫描的应用和局限性。
DOI：
10.1124/dmd.120.000264
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发表时间：
2021-05
期刊：
影响因子：
--
通讯作者：
Weinshilboum RM
中科院分区：
文献类型：
--
作者：
Zhang L;Sarangi V;Ho MF;Moon I;Kalari KR;Wang L;Weinshilboum RM
溶质载体有机阴离子转运蛋白家族成员1B1（solute carrier organic anion transporter family member 1B1）是一种重要的跨膜肝摄取转运蛋白。SLCO1B1基因中的遗传变异与蛋白质折叠改变相关，导致蛋白质降解和转运蛋白活性降低。药物基因组的下一代测序（NGS）正越来越多地应用于将药物反应的变化与遗传序列变异相关联。然而，很难使用“一次一个”功能系统将未知意义的变异与功能表型联系起来。使用“基于着陆垫细胞的系统”的深度突变扫描（DMS）是一种高通量技术，其设计用于以并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分析了从外显子组聚集联盟项目中获得的SLCO1B1 ORF中的137个错义变体。将含有这些变体的ORF与绿色荧光蛋白融合并整合到“着陆垫”细胞中。进行荧光激活的细胞分选，以基于指示单细胞水平的蛋白质表达的荧光读数将细胞分成四组。然后进行NGS，并使用SLCO1B1变体频率来确定蛋白质丰度。我们发现，6个以前没有功能特征的变体显示不到25%，另外12个显示约50%的野生型蛋白表达。然后通过转运蛋白研究对这些结果进行功能验证。通过DMS鉴定的严重破坏性变体可能与SLCO1B1依赖性药物转运具有临床相关性，但我们需要谨慎，因为鉴定的相对少量的严重破坏性变体引起了关于DMS应用于内源性膜蛋白（如有机阴离子转运蛋白1B1）的问题。转运蛋白基因中大量开放阅读框（ORF）“意义不明的变体”（VUS）的功能意义尚未得到表征。本研究应用深度突变扫描来确定在SLCO1B1（溶质载体有机阴离子转运蛋白家族成员1B1）的ORF中观察到的VUS的功能效应。几个严重破坏性的变种被确定，研究和验证。这些观察结果对内在膜蛋白的深度突变扫描的应用和SLCO1B1转运的药物和内源性化合物的临床效果都有影响。
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. 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(solute 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.
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DOI：
10.1016/j.bbrc.2004.10.040
发表时间：
2004-12-10
影响因子：
3.1
作者：
Wang, LW;Yee, VC;Weinshilboum, RM
通讯作者：
Weinshilboum, RM
影响因子：
6.7
作者：
Niemi, M.
通讯作者：
Niemi, M.
影响因子：
48
作者：
Fowler, Douglas M.;Fields, Stanley
通讯作者：
Fields, Stanley
影响因子：
3.9
作者：
Devarajan, Sandhya;Moon, Irene;Reid, Joel M.
通讯作者：
Reid, Joel M.
影响因子：
3.8
作者：
Dudenkov, Tanda M.;Ingle, James N.;Weinshilboum, Richard M.
通讯作者：
Weinshilboum, Richard M.