Grampa Valentina, Delous Marion, Zaidan Mohamad, Odye Gweltas, Thomas Sophie, Elkhartoufi Nadia, Filhol Emilie, Niel Olivier, Silbermann Flora, Lebreton Corinne, Collardeau-Frachon Sophie, Rouvet Isabelle, Alessandri Jean-Luc, Devisme Louise, Dieux-Coeslier Anne, Cordier Marie-Pierre, Capri Yline, Khung-Savatovsky Suonavy, Sigaudy Sabine, Salomon Rémi, Antignac Corinne, Gubler Marie-Claire, Benmerah Alexandre, Terzi Fabiola, Attié-Bitach Tania, Jeanpierre Cécile, Saunier Sophie
INSERM UMR1163, Laboratory of Inherited Kidney Diseases, Necker-Enfants Malades Hospital, Paris, France.
Paris Descartes-Sorbonne Paris Cité University, Imagine Institute, Paris, France.
PLoS Genet. 2016 Mar 11;12(3):e1005894. doi: 10.1371/journal.pgen.1005894. eCollection 2016 Mar.
Ciliopathies are a group of genetic multi-systemic disorders related to dysfunction of the primary cilium, a sensory organelle present at the cell surface that regulates key signaling pathways during development and tissue homeostasis. In order to identify novel genes whose mutations would cause severe developmental ciliopathies, >500 patients/fetuses were analyzed by a targeted high throughput sequencing approach allowing exome sequencing of >1200 ciliary genes. NEK8/NPHP9 mutations were identified in five cases with severe overlapping phenotypes including renal cystic dysplasia/hypodysplasia, situs inversus, cardiopathy with hypertrophic septum and bile duct paucity. These cases highlight a genotype-phenotype correlation, with missense and nonsense mutations associated with hypodysplasia and enlarged cystic organs, respectively. Functional analyses of NEK8 mutations in patient fibroblasts and mIMCD3 cells showed that these mutations differentially affect ciliogenesis, proliferation/apoptosis/DNA damage response, as well as epithelial morphogenesis. Notably, missense mutations exacerbated some of the defects due to NEK8 loss of function, highlighting their likely gain-of-function effect. We also showed that NEK8 missense and loss-of-function mutations differentially affect the regulation of the main Hippo signaling effector, YAP, as well as the expression of its target genes in patient fibroblasts and renal cells. YAP imbalance was also observed in enlarged spheroids of Nek8-invalidated renal epithelial cells grown in 3D culture, as well as in cystic kidneys of Jck mice. Moreover, co-injection of nek8 MO with WT or mutated NEK8-GFP RNA in zebrafish embryos led to shortened dorsally curved body axis, similar to embryos injected with human YAP RNA. Finally, treatment with Verteporfin, an inhibitor of YAP transcriptional activity, partially rescued the 3D spheroid defects of Nek8-invalidated cells and the abnormalities of NEK8-overexpressing zebrafish embryos. Altogether, our study demonstrates that NEK8 human mutations cause major organ developmental defects due to altered ciliogenesis and cell differentiation/proliferation through deregulation of the Hippo pathway.
纤毛病是一组与初级纤毛功能障碍相关的遗传性多系统疾病，初级纤毛是一种位于细胞表面的感觉细胞器，在发育和组织稳态过程中调节关键信号通路。为了鉴定其突变会导致严重发育性纤毛病的新基因，采用靶向高通量测序方法对500多名患者/胎儿进行了分析，该方法允许对1200多个纤毛基因进行外显子组测序。在5例具有严重重叠表型的病例中鉴定出NEK8/NPHP9突变，这些表型包括肾囊性发育不良/发育不全、内脏反位、肥厚性间隔心肌病和胆管稀少。这些病例突出了基因型-表型相关性，错义突变和无义突变分别与发育不全和囊性器官增大相关。对患者成纤维细胞和mIMCD3细胞中NEK8突变的功能分析表明，这些突变对纤毛发生、增殖/凋亡/DNA损伤反应以及上皮形态发生有不同影响。值得注意的是，错义突变加剧了一些由于NEK8功能丧失导致的缺陷，突出了它们可能的功能获得效应。我们还表明，NEK8错义突变和功能丧失突变对患者成纤维细胞和肾细胞中主要Hippo信号效应器YAP的调节以及其靶基因的表达有不同影响。在3D培养中生长的Nek8缺失的肾上皮细胞的增大球体以及Jck小鼠的囊性肾脏中也观察到YAP失衡。此外，在斑马鱼胚胎中共注射nek8 MO与野生型或突变型NEK8-GFP RNA导致背侧弯曲体轴缩短，类似于注射人YAP RNA的胚胎。最后，用YAP转录活性抑制剂维替泊芬治疗部分挽救了Nek8缺失细胞的3D球体缺陷和NEK8过表达斑马鱼胚胎的异常。总之，我们的研究表明，人类NEK8突变通过Hippo通路失调导致纤毛发生和细胞分化/增殖改变，从而引起主要器官发育缺陷。
