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Kidney Week

Abstract: SA-OR097

The Pathogenic Role of NEK8 RCC1-Domain Mutations in Inversin Compartment Assembly

Session Information

Category: Genetic Diseases of the Kidneys

  • 1001 Genetic Diseases of the Kidneys: Cystic

Authors

  • Attia, Doaa A., Brighams and Women's Hospital/Harvard Medical School, Boston, Massachusetts, United States
  • Misra, Anjali, University College Cork, Cork, Ireland
  • Czarnecki, Peter G., Brigham and Women's Hospital, Boston, Massachusetts, United States
Background

The Inversin compartment (IC) is a protein module located at the proximal end of the primary cilium that consists of four known components, inversin (INVS), NEK8, ANKS6 and NPHP3. Missense mutations in inversin compartment genes, including NEK8, give rise to a wide spectrum of disease phenotypes, including Nephronophthisis (NPHP), polycystic kidney disease (PKD), as well as complex multiorgan malformation syndromes with embryonic lethality. In our previous work, we have shown that NEK8 kinase-domain mutations that specifically affect the phosphorylation mechanism give rise to cystic kidney phenotypes and randomization of embryonic L-/R-asymmetry determination. We have also shown that the NEK8 RCC1-domain is necessary for recruitment of NPHP3 to the ciliary inversin compartment. The mechanism how NEK8 RCC1-domain mutations and mutations in NPHP3 produce NPHP- and PKD-like phenotypes remains unexplained.

Methods

We cloned affinity-tagged versions of NEK8 and NPHP3 in expression vectors for transient expression in 293T cells and for stable lentiviral transduction in mIMCD3 cells. We knocked out NEK8 and NPHP3 genes in IMCD3 cells by CRISPR/Cas9. We introduced pathogenic mutations into the NEK8 ORF by fusion PCR. We analyzed protein expression levels by Western-Blot, protein localization to primary cilia by immunofluorescence and protein-protein interactions by immunoprecipitation and Western-Blot.

Results

We found that pathologic missense mutations in the NEK8 RCC1 domain fall into three categories: (1) Mutations that destabilize NEK8 protein lead to reduced or absent expression and no detectable localization to the cilium; (2) Mutations that produce soluble and detectable levels of NEK8 protein, but fail to properly localize to the primary cilium; (3) Mutations that allow NEK8 protein to localize to the cilium, but have reduced affinity to NPHP3 binding. While mutations of categories (1) and (2) correlate with NPHP phenotypes, mutations of category (3) correlate with PKD phenotypes.

Conclusion

We demonstrate that hierarchical defects in inversin compartment assembly correlate with the phenotypic spectrum of kidney disease. Reduced NEK8 and NPHP3 localization leads to NPHP-like disease, whereas proper NEK8 localization and reduced NPHP3 affinity leads to PKD.