Byrne Jane A, Strautnieks Sandra S, Ihrke Gudrun, Pagani Franco, Knisely A S, Linton Kenneth J, Mieli-Vergani Giorgina, Thompson Richard J
Division of Gene and Cell Based Therapy, King's College London School of Medicine, London, UK.
Hepatology. 2009 Feb;49(2):553-67. doi: 10.1002/hep.22683.
The gene encoding the human bile salt export pump (BSEP), ABCB11, is mutated in several forms of intrahepatic cholestasis. Here we classified the majority (63) of known ABCB11 missense mutations and 21 single-nucleotide polymorphisms (SNPs) to determine whether they caused abnormal ABCB11 pre-messenger RNA splicing, abnormal processing of BSEP protein, or alterations in BSEP protein function. Using an in vitro minigene system to analyze splicing events, we found reduced wild-type splicing for 20 mutations/SNPs, with normal mRNA levels reduced to 5% or less in eight cases. The common ABCB11 missense mutation encoding D482G enhanced aberrant splicing, whereas the common SNP A1028A promoted exon skipping. Addition of exogenous splicing factors modulated several splicing defects. Of the mutants expressed in vitro in CHO-K1 cells, most appeared to be retained in the endoplasmic reticulum and degraded. A minority had BSEP levels similar to wild-type. The SNP variant A444 had reduced levels of protein compared with V444. Treatment with glycerol and incubation at reduced temperature overcame processing defects for several mutants, including E297G. Taurocholate transport by two assessed mutants, N490D and A570T, was reduced compared with wild-type.
This work is a comprehensive analysis of 80% of ABCB11 missense mutations and single-nucleotide polymorphisms at pre-mRNA splicing and protein processing/functional levels. We show that aberrant pre-mRNA splicing occurs in a considerable number of cases, leading to reduced levels of normal mRNA. Thus, primary defects at either the protein or the mRNA level (or both) contribute significantly to BSEP deficiency. These results will help to develop mutation-specific therapies for children and adults suffering from intrahepatic cholestasis due to BSEP deficiency.
编码人类胆盐输出泵（BSEP）的基因ABCB11在几种肝内胆汁淤积症中发生突变。在此，我们对大多数（63个）已知的ABCB11错义突变和21个单核苷酸多态性（SNP）进行分类，以确定它们是否导致ABCB11前体信使RNA剪接异常、BSEP蛋白加工异常或BSEP蛋白功能改变。利用体外小基因系统分析剪接事件，我们发现20个突变/SNP的野生型剪接受损，8例中正常mRNA水平降至5%或更低。编码D482G的常见ABCB11错义突变增强了异常剪接，而常见SNP A1028A促进外显子跳跃。添加外源性剪接因子可调节多种剪接缺陷。在CHO-K1细胞中体外表达的突变体中，大多数似乎保留在内质网中并被降解。少数突变体的BSEP水平与野生型相似。与V444相比，SNP变体A444的蛋白水平降低。用甘油处理并在低温下孵育可克服包括E297G在内的几种突变体的加工缺陷。与野生型相比，两个评估的突变体N490D和A570T的牛磺胆酸盐转运减少。
这项工作是对80%的ABCB11错义突变和单核苷酸多态性在mRNA前体剪接以及蛋白质加工/功能水平上的全面分析。我们表明，相当数量的病例中发生了异常的mRNA前体剪接，导致正常mRNA水平降低。因此，蛋白质或mRNA水平（或两者）的原发性缺陷对BSEP缺乏有显著影响。这些结果将有助于为因BSEP缺乏而患有肝内胆汁淤积症的儿童和成人开发针对特定突变的治疗方法。
