Novel NEK8 Mutations Cause Severe Syndromic Renal Cystic Dysplasia through YAP Dysregulation

Abstract

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.

Hippo‐Yap/Taz signaling: Complex network interactions and impact in epithelial cell behavior

“…There is evidence that cytoplasmic sequestration of Yap/Taz regulates unique aspects of differentiation of the mature epithelium. This is well illustrated by their reported role in the formation of primary cilia or multicilia in epithelial progenitors during organogenesis (Grampa et al, 2016;Habbig et al, 2011;Hossain et al, 2007;M. Kim, Kim, Lee, Kim, & Lim, 2014).…”

Mechanisms for nonmitotic activation of Aurora-A at cilia

“…Studies addressing the mechanisms regulating assembly and disassembly typically identify Aurora A (AURKA) as a proximal component of the disassembly machinery, activated by interaction with a group of directly interacting partner proteins, and sometimes implicating its interactions with a tubulin deacetylase, HDAC6 [38] (Figure 1). More recently, other components of cilia disassembly mechanism have been defined, including signaling from PLK1 – another kinase best known for function in mitosis – and the microtubule-associated kinesin motor KIF2A [45], the NEK2 kinase with an alternative kinesin, KIF24 [46], and other pathways (e.g., NEK8 [47, 48]). For PLK1 and NEK8, potential mechanisms for crosstalk with AURKA have been shown.…”

“…Grampa et al found that mutations in Nek8 genes are associated with severe renal cystic dysplasia phenotype in human patients and in mouse models, in which Nek8 influences ciliogenesis and cell cycle, possibly through regulation of the nucleocytoplasmic shuttle of YAP, a main Hippo pathway effector. Interestingly, while Nek8 shRNA knockdown or overexpression of wild type Nek8 did not affect ciliogenesis, overexpression of cystic renal dysplasia-associated mutants of Nek8 (G580S and R602W) resulted in significant loss of cilia in cultured cells [48]. In patient fibroblasts with such mutants, there was increased YAP activation and nuclear localization.…”

Zebrafish: A Functional Refuge at the End of an Odyssey

“…2,3 Grampa et al 4 evaluated genomic material from 342 living patients and 200 fetal losses with cystic kidney disease using a targeted exome strategy where 1222 genes associated with cilia function were sequenced. One known cilia protein encoding gene, NEK8/ NPHP9, encodes a serine/threonine kinase located at the inversin compartment of the cilial axoneme, distal to the transition zone, that was hypothesized to regulate the Hippo pathway.…”