反义寡核苷酸可挽救 ABCB11 基因中的内含子剪接变异，该变异导致进行性家族性肝内胆汁淤积症 2 型。

Antisense oligonucleotides rescue an intronic splicing variant in the ABCB11 gene that causes progressive familial intrahepatic cholestasis type 2.

机构信息

Department of Gastroenterology, Children's Hospital of Nanjing Medical University, Nanjing, China.

Department of Pathology, Children's Hospital of Nanjing Medical University, Nanjing, China.

出版信息

Dig Liver Dis. 2022 Nov;54(11):1541-1547. doi: 10.1016/j.dld.2022.04.002. Epub 2022 Apr 27.

DOI:10.1016/j.dld.2022.04.002

PMID:35490150

Abstract

BACKGROUND

Progressive familial intrahepatic cholestasis type 2 (PFIC2) is a rare disorder caused by variants in the ABCB11 gene encoding the bile salt export pump (BSEP). We investigated the molecular defect in a PFIC2 infant and rescued the splicing defect with antisense oligonucleotides (ASOs).

METHODS

Whole-exome sequencing (WES) revealed compound heterozygous variants in the ABCB11 gene in a PFIC2 patient. Liver biopsy was immunostained for BSEP. The splicing effect of the candidate variants was investigated by minigene assay. ASOs were designed to rescue aberrant splicing.

RESULTS

A Chinese girl of two nonconsanguineous healthy parents suffered from low glutamyl transpeptidase cholestasis and showed no response to the ursodeoxycholic acid. WES revealed that the patient was compound heterozygous for two novel variants in the ABCB11 gene: c.76+29T>G and c.390-2A>G. Liver immunohistochemistry showed the absence of BSEP. The variant c.76+29T>G was confirmed to retain 42 bp in the mature mRNA. The variant c.390-2A>G was confirmed to cause exon 6 skipping. We designed two ASOs and identified one of them that efficiently induced pseudoexon exclusion.

CONCLUSION

We reported two novel variants of the ABCB11 gene, c.76+29T>G and c.390-2A>G, in a PFIC2 infant, thereby expanding the genotype of PFIC2. Our findings provide evidence for ASOs as a therapeutic approach for PFIC2 patients carrying intronic variants.

摘要

背景

进行性家族性肝内胆汁淤积症 2 型（PFIC2）是一种由编码胆汁盐输出泵（BSEP）的 ABCB11 基因突变引起的罕见疾病。我们研究了一名 PFIC2 婴儿的分子缺陷，并使用反义寡核苷酸（ASO）来纠正剪接缺陷。

方法

全外显子组测序（WES）显示 PFIC2 患者的 ABCB11 基因存在复合杂合变异。对肝活检进行 BSEP 免疫染色。通过迷你基因试验研究候选变异的剪接效应。设计 ASO 以纠正异常剪接。

结果

两名非近亲健康父母所生的一名中国女孩患有低谷氨酰转肽酶胆汁淤积症，且对熊去氧胆酸无反应。WES 显示患者的 ABCB11 基因存在两个新的复合杂合变异：c.76+29T>G 和 c.390-2A>G。肝免疫组化显示 BSEP 缺失。变异 c.76+29T>G 被确认为在成熟 mRNA 中保留了 42bp。变异 c.390-2A>G 被确认为导致外显子 6 缺失。我们设计了两种 ASO，并确定了其中一种能有效诱导假外显子排除。