Autosomal dominant polycystic kidney disease (ADPKD) resulting from pathogenic variants in PKD1 and PKD2 is the most common form of PKD, but other genetic causes tied to primary cilia function have been identified. Biallelic pathogenic variants in the serine/threonine kinase NEK8 cause a syndromic ciliopathy with extra-kidney manifestations. Here we identify NEK8 as a disease gene for ADPKD in 12 families. Clinical evaluation was combined with functional studies using fibroblasts and tubuloids from affected individuals. Nek8 knockout mouse kidney epithelial (IMCD3) cells transfected with wild type or variant NEK8 were further used to study ciliogenesis, ciliary trafficking, kinase function, and DNA damage responses. Twenty-one affected monoallelic individuals uniformly exhibited cystic kidney disease (mostly neonatal) without consistent extra-kidney manifestations. Recurrent de novo mutations of the NEK8 missense variant p.Arg45Trp, including mosaicism, were seen in ten families. Missense variants elsewhere within the kinase domain (p.Ile150Met and p.Lys157Gln) were also identified. Functional studies demonstrated normal localization of the NEK8 protein to the proximal cilium and no consistent cilia formation defects in patient-derived cells. NEK8-wild type protein and all variant forms of the protein expressed in Nek8 knockout IMCD3 cells were localized to cilia and supported ciliogenesis. However, Nek8 knockout IMCD3 cells expressing NEK8-p.Arg45Trp and NEK8-p.Lys157Gln showed significantly decreased polycystin-2 but normal ANKS6 localization in cilia. Moreover, p.Arg45Trp NEK8 exhibited reduced kinase activity in vitro. In patient derived tubuloids and IMCD3 cells expressing NEK8-p.Arg45Trp, DNA damage signaling was increased compared to healthy passage-matched controls. Thus, we propose a dominant-negative effect for specific heterozygous missense variants in the NEK8 kinase domain as a new cause of PKD.

Birmingham Women's and Children's National Health Services West Midlands Birmingham UK

Department of Biochemistry and Molecular Biology Mayo Clinic Rochester Minnesota USA

Department of Biochemistry and Molecular Biology Mayo Clinic Rochester Minnesota USA; Division of Nephrology and Hypertension Mayo Clinic Rochester Minnesota USA

Department of Clinical Genetics Erasmus University Medical Center Rotterdam the Netherlands

Department of Clinical Genetics Odense University Hospital Odense Denmark; Department of Clinical Research University of Southern Denmark Odense Denmark

Department of Genetics and Biochemistry Clemson University Clemson South Carolina USA

Department of Genetics University Medical Center Utrecht Utrecht the Netherlands

Department of Molecular Genetics University of Toronto Toronto Ontario Canada; Pathology and Laboratory Medicine Mount Sinai Hospital Toronto Ontario Canada; Lunenfeld Tanenbaum Research Institute Toronto Ontario Canada

Department of Nephrology and Hypertension Regenerative Medicine Center Utrecht University Medical Center Utrecht Utrecht the Netherlands

Department of Nephrology and Hypertension University Medical Centre Utrecht Utrecht the Netherlands

Department of Nephrology and Hypertension University Medical Centre Utrecht Utrecht the Netherlands; Hubrecht Institute for Developmental Biology and Stem Cell Research KNAW Utrecht the Netherlands

Department of Pediatric Nephrology Erasmus University Medical Center Sophia Children's Hospital Rotterdam the Netherlands

Department of Pediatric Nephrology Wilhelmina Children's Hospital Utrecht the Netherlands

Department of Pediatrics 2nd Faculty of Medicine Charles University Prague Czech Republic; Department of Pediatrics University Hospital Ostrava Ostrava Czech Republic; Faculty of Medicine University of Ostrava Ostrava Czech Republic

Division of Nephrology and Hypertension Cincinnati Children's Hospital Medical Center Cincinnati Ohio USA

Division of Nephrology and Hypertension Mayo Clinic Rochester Minnesota USA

Division of Nephrology and Hypertension Mayo Clinic Rochester Minnesota USA; Division of Pediatric Nephrology and Hypertension Mayo Clinic Rochester Minnesota USA

Division of Nephrology Cliniques Universitaires Saint Luc Brussels Belgium; Recherche Expérimentale et Clinique UCLouvain Brussels Belgium

Fred A Litwin Family Centre in Genetic Medicine University Health Network and Mount Sinai Hospital Toronto Ontario Canada; Department of Medicine University of Toronto Toronto Ontario Canada

Fred A Litwin Family Centre in Genetic Medicine University Health Network and Mount Sinai Hospital Toronto Ontario Canada; Department of Molecular Genetics University of Toronto Toronto Ontario Canada

Fundeni Clinical Institute Bucharest Romania

Institute of Biology and Medical Genetics 1st Faculty of Medicine Charles University and General University Hospital Prague Czech Republic

Institute Pathology and Genetic Center of Human Genetics Charleroi Belgium

Newcastle University Translational and Clinical Research Institute Newcastle upon Tyne UK

Newcastle University Translational and Clinical Research Institute Newcastle upon Tyne UK; Renal Services The Newcastle upon Tyne Hospitals NHS Foundation Trust Newcastle UK; National Institute for Health and Care Research Biomedical Research Centre Newcastle UK

Pathology and Laboratory Medicine Mount Sinai Hospital Toronto Ontario Canada; Lunenfeld Tanenbaum Research Institute Toronto Ontario Canada

Research Division Greenwood Genetic Center Greenwood South Carolina USA

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Torres VE, Harris PC, Pirson Y. Autosomal dominant polycystic kidney disease. The Lancet. 2007;369(9569):1287–1301. doi:10.1016/S0140-6736(07)60601-1 PubMed DOI

Shaw C, Simms RJ, Pitcher D, Sandford R. Epidemiology of patients in England and Wales with autosomal dominant polycystic kidney disease and end-stage renal failure. Nephrol Dial Transplant. 2014;29(10):1910–1918. doi:10.1093/ndt/gfu087 PubMed DOI

Besse W, Dong K, Choi J, et al. Isolated polycystic liver disease genes define effectors of polycystin-1 function. J Clin Invest. 2017;127(5):1772–1785. doi:10.1172/JCI90129 PubMed DOI PMC

Besse W, Chang AR, Luo JZ, et al. ALG9 Mutation Carriers Develop Kidney and Liver Cysts. J Am Soc Nephrol. 2019;30(11):2091–2102. doi:10.1681/ASN.2019030298 PubMed DOI PMC

Senum SR, Li YSM, Benson KA, et al. Monoallelic IFT140 pathogenic variants are an important cause of the autosomal dominant polycystic kidney-spectrum phenotype. Am J Hum Genet. 2022;109(1):136–156. doi:10.1016/j.ajhg.2021.11.016 PubMed DOI PMC

Cornec-Le Gall E, Olson RJ, Besse W, et al. Monoallelic Mutations to DNAJB11 Cause Atypical Autosomal-Dominant Polycystic Kidney Disease. Am J Hum Genet. 2018;102(5):832–844. doi:10.1016/j.ajhg.2018.03.013 PubMed DOI PMC

Porath B, Gainullin VG, Cornec-Le Gall E, et al. Mutations in GANAB, Encoding the Glucosidase IIα Subunit, Cause Autosomal-Dominant Polycystic Kidney and Liver Disease. Am J Hum Genet. 2016;98(6):1193–1207. doi:10.1016/j.ajhg.2016.05.004 PubMed DOI PMC

Lemoine H, Raud L, Foulquier F, et al. Monoallelic pathogenic ALG5 variants cause atypical polycystic kidney disease and interstitial fibrosis. Am J Hum Genet. 2022;109(8):1484–1499. doi:10.1016/J.AJHG.2022.06.013 PubMed DOI PMC

Waters AM, Beales PL. Ciliopathies: An expanding disease spectrum. Pediatric Nephrology. 2011;26(7):1039–1056. doi:10.1007/s00467-010-1731-7 PubMed DOI PMC

Hildebrandt F, Benzing T, Katsanis N. Ciliopathies. New England Journal of Medicine. 2011;364(16):1533–1543. doi:10.1056/nejmra1010172 PubMed DOI PMC

Chaki M, Airik R, Ghosh AK, et al. Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling. Cell. 2012;150(3):533–548. doi:10.1016/j.cell.2012.06.028 PubMed DOI PMC

Ateş EA, Turkyilmaz A, Delil K, et al. Biallelic Mutations in DNAJB11 Are Associated with Prenatal Polycystic Kidney Disease in a Turkish Family. Vol 12. Mol Syndromol; 2021:179–185. doi:10.1159/000513611 PubMed DOI PMC

Jordan P, Arrondel C, Bessières B, et al. Bi-allelic pathogenic variations in DNAJB11 cause Ivemark II syndrome, a renal-hepatic-pancreatic dysplasia. Kidney Int. 2021;99(2):405–409. doi:10.1016/J.KINT.2020.09.029 PubMed DOI

Grampa V, Delous M, Zaidan M, et al. Novel NEK8 Mutations Cause Severe Syndromic Renal Cystic Dysplasia through YAP Dysregulation. PLoS Genet. 2016;12(3):e1005894. doi:10.1371/journal.pgen.1005894 PubMed DOI PMC

Frank V, Habbig S, Bartram MP, et al. Mutations in NEK8 link multiple organ dysplasia with altered Hippo signalling and increased c-MYC expression. Hum Mol Genet. 2013;22(11):2177–2185. doi:10.1093/hmg/ddt070 PubMed DOI

Hoff S, Halbritter J, Epting D, et al. ANKS6 is a central component of a nephronophthisis module linking NEK8 to INVS and NPHP3. Nat Genet. 2013;45(8):951–956. doi:10.1038/ng.2681 PubMed DOI PMC

Sohara E, Luo Y, Zhang J, Manning DK, Beier DR, Zhou J. Nek8 regulates the expression and localization of polycystin-1 and polycystin-2. Journal of the American Society of Nephrology. 2008;19(3):469–476. doi:10.1681/ASN.2006090985 PubMed DOI PMC

Sobreira N, Schiettecatte F, Valle D, Hamosh A. GeneMatcher: A Matching Tool for Connecting Investigators with an Interest in the Same Gene. Hum Mutat. 2015;36(10):928–930. doi:10.1002/humu.22844 PubMed DOI PMC

Smedley D, Smith KR, Martin A, et al. 100,000 Genomes Pilot on Rare-Disease Diagnosis in Health Care - Preliminary Report. N Engl J Med. 2021;385(20):1868–1880. doi:10.1056/NEJMOA2035790 PubMed DOI PMC

Varadi M, Anyango S, Deshpande M, et al. AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res. 2022;50(D1):D439–D444. doi:10.1093/NAR/GKAB1061 PubMed DOI PMC

Jumper J, Evans R, Pritzel A, et al. Highly accurate protein structure prediction with AlphaFold. Nature 2021 596:7873. 2021;596(7873):583–589. doi:10.1038/s41586-021-03819-2 PubMed DOI PMC

Mariani V, Biasini M, Barbato A, Schwede T. lDDT: a local superposition-free score for comparing protein structures and models using distance difference tests. Bioinformatics. 2013;29(21):2722. doi:10.1093/BIOINFORMATICS/BTT473 PubMed DOI PMC

Rentzsch P, Schubach M, Shendure J, Kircher M. CADD-Splice—improving genome-wide variant effect prediction using deep learning-derived splice scores. Genome Med. 2021;13(1). doi:10.1186/s13073-021-00835-9 PubMed DOI PMC

Wiel L, Baakman C, Gilissen D, Veltman JA, Vriend G, Gilissen C. MetaDome: Pathogenicity analysis of genetic variants through aggregation of homologous human protein domains. Hum Mutat. 2019;40(8):1030–1038. doi:10.1002/humu.23798 PubMed DOI PMC

Manning DK, Sergeev M, van Heesbeen RG, et al. Loss of the ciliary kinase Nek8 causes left-right asymmetry defects. J Am Soc Nephrol. 2013;24(1):100–112. doi:10.1681/ASN.2012050490 PubMed DOI PMC

Johannessen CM, Boehm JS, Kim SY, et al. COT/MAP3K8 drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature. 2010;468(7326):968. doi:10.1038/NATURE09627 PubMed DOI PMC

Chen C, Xu Q, Zhang Y, et al. Ciliopathy protein HYLS1 coordinates the biogenesis and signaling of primary cilia by activating the ciliary lipid kinase PIPKIγ. Sci Adv. 2021;7(26). doi:10.1126/SCIADV.ABE3401 PubMed DOI PMC

Obeidova L, Seeman T, Fencl F, et al. Results of targeted next-generation sequencing in children with cystic kidney diseases often change the clinical diagnosis. PLoS One. 2020;15(6):e0235071. PubMed PMC

Karczewski KJ, Francioli LC, Tiao G, et al. The mutational constraint spectrum quantified from variation in 141,456 humans. Nature. 2020;581(7809):434–443. doi:10.1038/s41586-020-2308-7 PubMed DOI PMC

Holland PM, Milne A, Garka K, et al. Purification, cloning, and characterization of Nek8, a novel nima-related kinase, and its candidate substrate Bicd2. Journal of Biological Chemistry. 2002;277(18):16229–16240. doi:10.1074/jbc.M108662200 PubMed DOI

Tsai J, Gerstein M. Calculations of protein volumes: Sensitivity analysis and parameters database. Bioinformatics. 2002;18(7):985–995. doi:10.1093/bioinformatics/18.7.985 PubMed DOI

Czarnecki PG, Gabriel GC, Manning DK, et al. ANKS6 is the critical activator of NEK8 kinase in embryonic situs determination and organ patterning. Nat Commun. 2015;6. doi:10.1038/ncomms7023 PubMed DOI PMC

Kinoshita E, Kinoshita-Kikuta E, Koike T. Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE. Nat Protoc. 2009;4(10):1513–1521. doi:10.1038/NPROT.2009.154 PubMed DOI

Helt CE, Cliby WA, Keng PC, Bambara RA, O’Reilly MA. Ataxia telangiectasia mutated (ATM) and ATM and Rad3-related protein exhibit selective target specificities in response to different forms of DNA damage. J Biol Chem. 2005;280(2):1186–1192. doi:10.1074/JBC.M410873200 PubMed DOI

Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem. 1998;273(10):5858–5868. doi:10.1074/JBC.273.10.5858 PubMed DOI

Ward IM, Chen J. Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress. J Biol Chem. 2001;276(51):47759–47762. doi:10.1074/JBC.C100569200 PubMed DOI

Choi HJC, Lin JR, Vannier JB, et al. NEK8 Links the ATR-Regulated Replication Stress Response and S Phase CDK Activity to Renal Ciliopathies. Mol Cell. 2013;51(4):423–439. doi:10.1016/j.molcel.2013.08.006 PubMed DOI PMC

Ali H, Al-Mulla F, Hussain N, et al. PKD1 Duplicated regions limit clinical Utility of Whole Exome Sequencing for Genetic Diagnosis of Autosomal Dominant Polycystic Kidney Disease. Sci Rep. 2019;9(1):4141. doi:10.1038/s41598-019-40761-w PubMed DOI PMC

Rajagopalan R, Grochowski CM, Gilbert MA, et al. Compound heterozygous mutations in NEK8 in siblings with end-stage renal disease with hepatic and cardiac anomalies. Am J Med Genet A. 2016;170(3):750–753. doi:10.1002/AJMG.A.37512 PubMed DOI

Otto EA, Trapp ML, Schultheiss UT, Helou J, Quarmby LM, Hildebrandt F. NEK8 mutations affect ciliary and centrosomal localization and may cause nephronophthisis. J Am Soc Nephrol. 2008;19(3):587–592. doi:10.1681/ASN.2007040490 PubMed DOI PMC

Domingo-Gallego A, Pybus M, Bullich G, et al. Clinical utility of genetic testing in early-onset kidney disease: seven genes are the main players. Nephrology, dialysis, transplantation. 2022;37(4):687–696. doi:10.1093/NDT/GFAB019 PubMed DOI

Bergmann C. ARPKD and early manifestations of ADPKD: the original polycystic kidney disease and phenocopies. Pediatr Nephrol. 2015;30(1):15–30. doi:10.1007/s00467-013-2706-2 PubMed DOI PMC

Mehawej C, Chouery E, Ghabril R, Tokajian S, Megarbane A. NEK8-Associated Nephropathies: Do Autosomal Dominant Forms Exist? Nephron. Published online 2022. doi:10.1159/000526841 PubMed DOI

Kent WJ, Sugnet CW, Furey TS, et al. The human genome browser at UCSC. Genome Res. 2002;12(6):996–1006. doi:10.1101/GR.229102 PubMed DOI PMC

Cooper DN, Mort M, Stenson PD, Ball EV., Chuzhanova NA. Methylation-mediated deamination of 5-methylcytosine appears to give rise to mutations causing human inherited disease in CpNpG trinucleotides, as well as in CpG dinucleotides. Hum Genomics. 2010;4(6):406–410. doi:10.1186/1479-7364-4-6-406 PubMed DOI PMC