Missense Mutations and Single Nucleotide Polymorphisms in ABCB11 Impair Bile Salt Export Pump Processing and Function or Disrupt Pre-Messenger RNA Splicing
Missense Mutations and Single Nucleotide Polymorphisms in ABCB11 Impair Bile Salt Export Pump Processing and Function or Disrupt Pre-Messenger RNA Splicing
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DOI：
10.1002/hep.22683
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发表时间：
2009-02-01
期刊：
影响因子：
13.5
通讯作者：
Thompson, Richard J.
中科院分区：
文献类型：
--
作者：
Byrne, Jane A.;Strautnieks, Sandra S.;Thompson, Richard J.
编码人类胆盐输出泵(BSEP)的基因ABCB11在几种形式的肝内胆汁淤积症中发生突变。在这里，我们对大多数(63)已知的ABCB11错义突变和21个单核苷酸多态(SNPs)进行分类，以确定它们是否导致ABCB11前信使RNA剪接异常、BSEP蛋白加工异常或BSEP蛋白功能改变。使用体外迷你基因系统分析剪接事件，我们发现20个突变/SNPs的野生型剪接减少，其中8例正常mRNA水平降至5%或更低。常见的编码D482G的ABCB11错义突变促进异常剪接，而共同的SNP A1028A促进外显子跳跃。外源剪接因子的加入调节了几种剪接缺陷。在CHO-K1细胞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。少数人的BSEP水平与野生型相似。与V444相比，SNP变异体A444的蛋白质水平降低。用甘油处理和低温孵育克服了包括E297G在内的几个突变体的加工缺陷。与野生型相比，两个被评估的突变体N490D和A570T的牛磺胆酸转运减少。结论：本工作是对80%的ABCB11错义突变和单核苷酸多态在前mRNA剪接和蛋白质加工/功能水平上的综合分析。我们发现，在相当多的情况下，会发生异常的前mRNA剪接，导致正常mRNA水平的降低。因此，蛋白质或信使核糖核酸水平的原发缺陷(或两者兼而有之)是BSEP缺陷的重要原因。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突变特异性治疗方法。(《肝病》2009；49：553-567。)
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. Conclusion: This work is a comprehensive analysis of 80% of ABCB11 Missense mutations and single-nudeotide 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. (HEPATOLOGY 2009;49:553-567.)