进行性家族性肝内胆汁淤积(PFIC)是一种常染色体隐性遗传性疾病,占儿童胆汁淤积的10%-15%,并可能导致婴儿残疾或死亡。有三种公认的PFIC类型(1-3),由ATP8B1,ABCB11和ABCB4基因突变引起。新报道了紧密连接蛋白2基因(TJP2)的双等位基因致病变异是PFIC4型的原因;然而,据报道,TJP2仅有数量有限的患者和无可争议的变异体,而PFIC4的潜在机制仍然知之甚少.探讨TJP2分析在PFIC1-3突变阴性的可疑PFIC患者中的诊断率。我们设计了一种基于多重聚合酶链反应的下一代测序方法来分析267例PFIC患者的TJP2基因变异,并鉴定了3例患者的双等位基因罕见变异,包括3例患者的3种已知致病变种和2种新型变种。通过使用CRISPR-cas9技术,我们证明TJP2c.1202A>G至少部分是通过增加TJP2蛋白的表达和核定位而致病的。通过小基因检测,我们发现TJP2c.2668-11A>G是通过诱导TJP2基因的异常剪接和过早截短的TJP2蛋白的翻译而产生的一种新的致病变异。此外,通过siRNA技术敲低TJP2蛋白导致细胞增殖抑制,诱导凋亡,分散的F-肌动蛋白,LO2和HepG2细胞中的微丝无序。TJP2敲低LO2细胞和HepG2细胞的全局基因表达谱鉴定了参与肌动蛋白细胞骨架调节的失调基因。微管细胞骨架基因在TJP2敲低细胞中显著下调。这项研究的结果表明,TJP2c.1202A>G和TJP2c.2668-11A>G是两个新的致病变体,细胞骨架相关的功能和途径可能是PFIC的潜在分子发病机制。
Progressive familial intrahepatic cholestasis (
PFIC) is an autosomal recessive inherited disease that accounts for 10%-15% childhood cholestasis and could lead to infant disability or death. There are three well-established types of
PFIC (1-3), caused by mutations in the ATP8B1, ABCB11, and ABCB4 genes. Biallelic pathogenic variants in the tight junction protein 2 gene (TJP2) were newly reported as a cause for
PFIC type 4; however, only a limited number of patients and undisputable variants have been reported for TJP2, and the underlying mechanism for
PFIC 4 remains poorly understood. To explore the diagnostic yield of TJP2 analysis in suspected
PFIC patients negative for the PFIC1-3 mutation, we designed a multiplex polymerase chain reaction-based next-generation sequencing method to analyze TJP2 gene variants in 267 PFIC patients and identified biallelic rare variants in three patients, including three known pathogenic variants and two novel variants in three patients. By using CRISPR-cas9 technology, we demonstrated that TJP2 c.1202A > G was pathogenic at least partially by increasing the expression and nuclear localization of TJP2 protein. With the minigene assay, we showed that TJP2 c.2668-11A > G was a new pathogenic variant by inducing abnormal splicing of TJP2 gene and translation of prematurely truncated TJP2 protein. Furthermore, knockdown of TJP2 protein by siRNA technology led to inhibition of cell proliferation, induction of apoptosis, dispersed F-actin, and disordered microfilaments in LO2 and HepG2celles. Global gene expression profiling of TJP2 knockdown LO2 cells and HepG2 cells identified the dysregulated genes involved in the regulation of actin cytoskeleton. Microtubule cytoskeleton genes were significantly downregulated in TJP2 knockdown cells. The results of this study demonstrate that TJP2 c.1202A > G and TJP2 c.2668-11A > G are two novel pathogenic variants and the cytoskeleton-related functions and pathways might be potential molecular pathogenesis for PFIC.