Université Paris-Saclay, Inserm, Physiopathogénèse et traitement des maladies du Foie, UMR_S 1193, Hepatinov, Orsay, France.
Pediatric Hepatology & Pediatric Liver Transplant Department, Centre de Référence de l'Atrésie des Voies Biliaires et des Cholestases Génétiques, Filière de Santé des Maladies Rares du Foie de l'enfant et de l'adulte, European Reference Network RARE-LIVER, Assistance Publique-Hôpitaux de Paris, Faculty of Medecine Paris-Saclay, CHU Bicêtre, Le Kremlin-Bicêtre, France.
Hepatology. 2021 Apr;73(4):1449-1463. doi: 10.1002/hep.31476.
Progressive familial intrahepatic cholestasis type 2 (PFIC2) is a severe hepatocellular cholestasis due to biallelic mutations in ABCB11 encoding the canalicular bile salt export pump (BSEP). Nonsense mutations are responsible for the most severe phenotypes. The aim was to assess the ability of drugs to induce readthrough of six nonsense mutations (p.Y354X, p.R415X, p.R470X, p.R1057X, p.R1090X, and p.E1302X) identified in patients with PFIC2.
The ability of G418, gentamicin, and PTC124 to induce readthrough was studied using a dual gene reporter system in NIH3T3 cells. The ability of gentamicin to induce readthrough and to lead to the expression of a full-length protein was studied in human embryonic kidney 293 (HEK293), HepG2, and Can 10 cells using immunodetection assays. The function of the gentamicin-induced full-length protein was studied by measuring the [ H]-taurocholate transcellular transport in stable Madin-Darby canine kidney clones co-expressing Na+-taurocholate co-transporting polypeptide (Ntcp). Combinations of gentamicin and chaperone drugs (ursodeoxycholic acid, 4-phenylbutyrate [4-PB]) were investigated. In NIH3T3, aminoglycosides significantly increased the readthrough level of all mutations studied, while PTC124 only slightly increased the readthrough of p.E1302X. Gentamicin induced a readthrough of p.R415X, p.R470X, p.R1057X, and p.R1090X in HEK293 cells. The resulting full-length proteins localized within the cytoplasm, except for Bsep , which was also detected at the plasma membrane of human embryonic kidney HEK293 and at the canalicular membrane of Can 10 and HepG2 cells. Additional treatment with 4-PB and ursodeoxycholic acid significantly increased the canalicular proportion of full-length Bsep protein in Can 10 cells. In Madin-Darby canine kidney clones, gentamicin induced a 40% increase of the Bsep [ H]-taurocholate transport, which was further increased with additional 4-PB treatment.
This study constitutes a proof of concept for readthrough therapy in selected patients with PFIC2 with nonsense mutations.
进行性家族性肝内胆汁淤积症 2 型（PFIC2）是一种严重的肝细胞胆汁淤积症，由 ABCB11 双等位基因突变引起，该基因编码胆小管胆汁盐输出泵（BSEP）。无义突变负责最严重的表型。目的是评估六种无义突变（p.Y354X、p.R415X、p.R470X、p.R1057X、p.R1090X 和 p.E1302X）在 PFIC2 患者中的药物诱导通读能力。
使用 NIH3T3 细胞中的双基因报告系统研究了 G418、庆大霉素和 PTC124 诱导通读的能力。使用免疫检测法在人胚肾 293（HEK293）、HepG2 和 Can 10 细胞中研究了庆大霉素诱导通读和导致全长蛋白表达的能力。通过测量稳定表达 Na+-牛磺胆酸钠共转运多肽（Ntcp）的 Madin-Darby 犬肾克隆中的 [ H]-牛磺胆酸的跨细胞转运来研究庆大霉素诱导的全长蛋白的功能。研究了庆大霉素与伴侣药物（熊去氧胆酸、4-苯基丁酸[4-PB]）的组合。在 NIH3T3 中，氨基糖苷类药物显著增加了所有研究突变的通读水平，而 PTC124 仅略微增加了 p.E1302X 的通读。庆大霉素诱导 HEK293 细胞中 p.R415X、p.R470X、p.R1057X 和 p.R1090X 的通读。除了 Bsep 外，产生的全长蛋白定位于细胞质内，Bsep 也在人胚肾 HEK293 的质膜和 Can 10 和 HepG2 细胞的胆小管膜上检测到。在用 4-PB 和熊去氧胆酸进一步处理后，Can 10 细胞中全长 Bsep 蛋白的胆小管比例显著增加。在 Madin-Darby 犬肾克隆中，庆大霉素诱导 Bsep [ H]-牛磺胆酸转运增加 40%，用额外的 4-PB 处理后进一步增加。
这项研究为具有无义突变的 PFIC2 患者的通读治疗提供了概念验证。
