Gene and Cell Therapy Laboratory, Vall d'Hebron Research Institute, Universitat Autònoma de Barcelona, Barcelona, Spain.
Congenital Coagulopathies Laboratory, Blood and Tissue Bank (BST), Barcelona, Spain.
J Thromb Haemost. 2017 Nov;15(11):2188-2197. doi: 10.1111/jth.13808. Epub 2017 Sep 25.
Essentials The Royal disease (RD) is a form of hemophilia B predicted to be caused by a splicing mutation. We generated an iPSC-based model of the disease allowing mechanistic studies at the RNA level. F9 mRNA analysis in iPSC-derived hepatocyte-like cells showed the predicted abnormal splicing. Mutated F9 mRNA level was very low but we also found traces of wild type transcripts.
Background The royal disease is a form of hemophilia B (HB) that affected many descendants of Queen Victoria in the 19th and 20th centuries. It was found to be caused by the mutation F9 c.278-3A>G. Objective To generate a physiological cell model of the disease and to study F9 expression at the RNA level. Methods Using fibroblasts from skin biopsies of a previously identified hemophilic patient bearing the F9 c.278-3A>G mutation and his mother, we generated induced pluripotent stem cells (iPSCs). Both the patient's and mother's iPSCs were differentiated into hepatocyte-like cells (HLCs) and their F9 mRNA was analyzed using next-generation sequencing (NGS). Results and Conclusion We demonstrated the previously predicted aberrant splicing of the F9 transcript as a result of an intronic nucleotide substitution leading to a frameshift and the generation of a premature termination codon (PTC). The F9 mRNA level in the patient's HLCs was significantly reduced compared with that of his mother, suggesting that mutated transcripts undergo nonsense-mediated decay (NMD), a cellular mechanism that degrades PTC-containing mRNAs. We also detected small proportions of correctly spliced transcripts in the patient's HLCs, which, combined with genetic variability in splicing and NMD machineries, could partially explain some clinical variability among affected members of the European royal families who had lifespans above the average. This work allowed the demonstration of the pathologic consequences of an intronic mutation in the F9 gene and represents the first bona fide cellular model of HB allowing the study of rare mutations at the RNA level.
生成该疾病的生理细胞模型，并研究 RNA 水平的 F9 表达。
使用先前鉴定的带有 F9 c.278-3A>G 突变的血友病患者及其母亲的皮肤活检纤维母细胞，生成诱导多能干细胞（iPSC）。患者和母亲的 iPSC 均分化为肝样细胞（HLC），并使用下一代测序（NGS）分析其 F9 mRNA。
我们证明了 F9 转录物的先前预测的异常剪接，这是由于内含子核苷酸取代导致移码和产生过早终止密码子（PTC）。与他的母亲相比，患者的 HLCs 中的 F9 mRNA 水平显着降低，这表明突变的转录本经历无意义介导的衰变（NMD），这是一种降解包含 PTC 的 mRNA 的细胞机制。我们还在患者的 HLCs 中检测到少量正确剪接的转录本，这与剪接和 NMD 机制中的遗传变异性相结合，可以部分解释在欧洲王室中受影响成员的一些临床变异性，这些成员的寿命超过平均水平。这项工作证明了 F9 基因内含子突变的病理后果，并代表了首个允许在 RNA 水平研究罕见突变的真正 HB 细胞模型。
