METHODS: 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.
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.
CONCLUSIONS: 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.
方法:ABCC2基因的靶向下一代测序用于鉴定欧洲受试者(n=143)中的新变体。使用了26种硅片工具来预测功能后果。对于生物验证,用从过表达新鉴定的ABCC2变体的细胞系制备的膜囊泡以及雌二醇β-葡糖苷酸和羧基二氯荧光素作为底物进行转运测定。
结果:确定了三个新颖的罕见ABCC2错义变体(W227R,K402T,V489F)。二十五个硅片工具预测W227R具有破坏性,一个可能具有破坏性。K402T和V489F以及常见的连锁变体V1188E/C1515Y不可能预测功能后果。体外表征显示W227R的功能增加,V489F和V1188E/C1515Y两种基板,而K402T功能仅在羧基二氯荧光素中增加。
结论:模拟工具无法准确预测ABCC2错义变体功能的底物依赖性增加。需要进行体外生物学研究以准确确定功能活性,以避免对药物治疗产生误导性后果。