Novel NEK8 Mutations Cause Severe Syndromic Renal Cystic Dysplasia through YAP Dysregulation.
Novel NEK8 Mutations Cause Severe Syndromic Renal Cystic Dysplasia through YAP Dysregulation.
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DOI：
10.1371/journal.pgen.1005894
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发表时间：
2016-03
期刊：
影响因子：
4.5
通讯作者：
Saunier S
中科院分区：
文献类型：
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作者：
Grampa V;Delous M;Zaidan M;Odye G;Thomas S;Elkhartoufi N;Filhol E;Niel O;Silbermann F;Lebreton C;Collardeau-Frachon S;Rouvet I;Alessandri JL;Devisme L;Dieux-Coeslier A;Cordier MP;Capri Y;Khung-Savatovsky S;Sigaudy S;Salomon R;Antignac C;Gubler MC;Benmerah A;Terzi F;Attié-Bitach T;Jeanpierre C;Saunier S
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，初级纤毛是存在于细胞表面的感觉细胞器，其在发育和组织稳态期间调节关键信号通路。为了鉴定其突变将引起严重发育性纤毛病的新基因，通过靶向高通量测序方法分析>500名患者/胎儿，所述靶向高通量测序方法允许>1200个纤毛基因的外显子组测序。NEK 8/NPHP 9突变在5例具有严重重叠表型的病例中被鉴定，包括肾囊性发育不良/发育不全、异位、心脏病伴肥厚性间隔和胆管缺乏。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良和囊性器官增大相关。患者成纤维细胞和mIMCD 3细胞中NEK 8突变的功能分析表明，这些突变差异影响纤毛发生，增殖/凋亡/DNA损伤反应，以及上皮形态发生。值得注意的是，错义突变加剧了由于NEK 8功能丧失而导致的一些缺陷，突出了它们可能的功能获得效应。我们还发现，NEK 8错义和功能丧失突变差异影响主要Hippo信号效应物雅普的调节，以及其靶基因在患者成纤维细胞和肾细胞中的表达。雅普不平衡还在3D培养中生长的Nek 8失效的肾上皮细胞的扩大球状体中以及在Jck小鼠的囊性肾中观察到。此外，在斑马鱼胚胎中共注射nek 8 MO与WT或突变的NEK 8-GFP RNA导致缩短背侧弯曲的体轴，类似于注射人雅普RNA的胚胎。最后，用Verteporfin（一种雅普转录活性抑制剂）治疗，部分挽救了Nek 8无效细胞的3D球状体缺陷和NEK 8过表达斑马鱼胚胎的异常。总之，我们的研究表明，NEK 8人类突变导致主要器官发育缺陷，由于改变纤毛发生和细胞分化/增殖通过河马途径的失调。纤毛病中突变的基因编码在初级纤毛中具有各种定位和功能的蛋白质。在这里，我们报告了肾脏囊性发育不良和相关纤毛病变缺陷患者的新NEK 8突变。NEK 8属于定义纤毛的反转蛋白区室的蛋白质复合物。它也是控制器官生长的Hippo信号通路的负调节剂。我们报告了患者的基因型-表型相关性。我们在功能上证明了两种类型的突变（错义与无义）差异影响纤毛发生，细胞凋亡和上皮形成。我们还表明，所有的突变导致通过核雅普不平衡的河马途径的失调，但这种不平衡的性质是不同的，根据突变的类型。我们证实了在Jck小鼠体内与Nek 8突变相关的Hippo通路的改变。值得注意的是，我们发现，形态发生缺陷中观察到的Nek 8敲低上皮细胞或斑马鱼胚胎被维替泊芬，特异性抑制剂的雅普转录活性，证明了在这些缺陷的发生雅普失调的病因作用。总而言之，这项研究将NEK 8突变与Hippo通路失调联系起来，并为了解患者多器官缺陷的变异性提供了分子线索。
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. Genes mutated in ciliopathies encode proteins with various localizations and functions at the primary cilium. Here we report novel NEK8 mutations in patients with renal cystic hypodysplasia and associated ciliopathy defects. NEK8 belongs to a protein complex defining the Inversin compartment of the cilium. It is also a negative regulator of the Hippo signaling pathway that controls organ growth. We report genotype-phenotype correlation in the patients. We functionally demonstrate that the two types of mutations (missense versus nonsense) differentially affect ciliogenesis, cell apoptosis and epithelialisation. We also show that all the mutations lead to dysregulation of the Hippo pathway through nuclear YAP imbalance but that the nature of this imbalance is different according to the type of mutation. We confirm alteration of the Hippo pathway associated with Nek8 mutation in vivo in Jck mice. Remarkably, we show that morphogenesis defects observed in Nek8 knockdown epithelial cells or zebrafish embryos are rescued by Verteporfin, a specific inhibitor of YAP transcriptional activity, demonstrating the causative role of YAP dysregulation in the occurrence of these defects. Altogether, this study links NEK8 mutations to dysregulation of the Hippo pathway and provide molecular clues to understand the variability of the multiorgan defects in the patients.