Certain heterozygous variants in the kinase domain of the serine/threonine kinase NEK8 can cause an autosomal dominant form of polycystic kidney disease

Abstract

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.

Keywords: NEK8; ciliopathy; kinase; polycystic kidney disease.

Figure 1.. Pedigrees and polycystic kidney images ∣

a. pedigrees of twelve families with heterozygous NEK8 variants. Solid symbols indicate affected individual with kidney disease. Arrow indicates proband. +/− indicates presence of heterozygous NEK8 variant. −/− indicate biallelic wildtype NEK8. Roman numerals indicate generation. MZ = monozygotic; d. death. b. MRI images of proband of Family 3 at age 16: enlarged kidney with multiple cysts, total kidney volume 1300 ml. c-d. Ultrasound of proband of Family 8: enlarged kidneys with extensive bilateral kidney cysts. At age 2 right kidney measured 13.6 cm and left kidney measured 13.3 cm (c), at age 7 right kidney: 17.5 cm and left kidney: 17.7 cm (d). e-g. CT analysis of Family 9: bilaterally enlarged kidneys with cysts. e. CT at 48 years of II-2, who is mosaic for the NEK8 variant, f-g. CT of proband IV-1 at 23 months (f) and at 5 years old (g). h. Pathology images of right kidney of proband of Family 4 at age 5 (20 x 11 x 10 cm, 1488 grams). i. Pathology images of right kidney of proband of Family 8 at 7 years old. j. Pathology images of left kidney of proband of Family 12 at age 4 (18 x 8 x 9.5 cm). k. Hematoxylin and eosin stain of proband of Family 8 showed multiple larger tubular cysts, with glomerular cysts also noted. l. Hematoxylin and eosin stain of proband of Family 12 shows multiple cysts both glomerular and tubular. Below the capsule cysts are smaller and they are larger at the corticomedullary junction. Some fibrosis is seen towards the medulla.

Figure 2.. Position of variants and domain organization of the NEK8 protein.

a. In red novel heterozygous variants described in this paper. In black variants previously published in biallelic cases. b. Protein Tolerance Landscape with identified variants and previously reported missense variants in kinase domain highlighted. Color coding indicates predicted impact of variant on protein function with blue =most tolerant, yellow= neutral, red=most detrimental. c. In silico predictions of the novel heterozygous NEK8 variants. d. Multiple sequence alignment of the human NEK8 full protein zoomed in on well conserved kinase domain. The amino acid positions of the heterozygous NEK8 variants are marked by the red arrows. e. Structural modeling of the NEK8 variants. Inset a. The predicted 3-dimentional structural model of human Serine/threonine-protein kinase NEK8 generated using AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk) and UniProtKB (https://www.uniprot.org/uniprot/) with associated codes: AF-Q86SG6-F1 and Q86SG6 respectively. The protein contains a protein kinase domain (red) and five ‘Regulator of Chromosome Condensation 1’ (RCC1) repeat domains. Inset b. Missense SNV c.133C>T p.Arg45Trp modelled with the most probable (26.5% likelihood) rotamer demonstrated. Inset c. Missense SNV c.450C>G p.Ile150Met modelled with the most probable (28.5% likelihood) rotamer demonstrated. Inset d. Positions of both variants in b. and c. highlighted in yellow (and shown as 1. and 2., respectively) to demonstrate their juxtaposition in adjacent alpha-helices. The third variant c.469A>G p.Lys157Gln could not be predicted due to a low confidence score, but appears to be in close proximity to the other variants as highlighted by the arrow (shown as 3.). A known active site (4., proton acceptor) and autophosphorylation residue (shown as 5.) are also in close proximity to the variants.

Figure 3.. Analyses of the function of the NEK8 variants

(a) Sanger sequencing results of cDNA PCR products showed a nucleotide insertion in Nek8 in the IMCD3 cell line, which causes a frameshift with early termination (45aa). (b-h) Nek8 knockout cells (−/−) were transfected with the empty vector (Mock) or one of the Myc-tagged NEK8 constructs, including the wild type (WT) and indicated single-point mutations. Forty-eight-hr post transfection, cells were treated with G418 (1 μg/mL) for one week. Cells survived the G418 selection were expanded, serum-starved for 24 h, and then subjected to the following analyses. Parental IMCD3 cells (+/+) were used as a positive control for endogenous Nek8, cells were harvested for immunoblotting (IB) with indicated antibodies, and α-tubulin was used as a loading control (b, h). (b) IB with a Myc or NEK8 antibody showed that all NEK8 proteins were expressed. (c-g) Cells were subjected to immunofluorescence (IF) microscopy using the indicated antibodies to visualize Myc-tagged NEK8 variants (c), ARL13B (d), ANKS6 (e), GPR161 (f), or PC2 (g) (scale bar, 2 μm). Primary cilia were labeled with an acetylated α-tubulin (Ac-tub) antibody. Data were collected from >100 cilia in each experimental group. Results from three independent experiments were statistically analyzed and plotted as mean ± SD. P values labeled in black were obtained by comparing to the group of Nek8^−/−, mock transfected cells (−/−, Mock). P values labelled in red were obtained by comparing to the Nek8^−/− cells stably expressing the wild type NEK8 (−/−, WT). (c) the NEK8 proteins with the various variants exhibited similar localization in cilia. (d, upper panel) Ciliogenesis was quantified by counting the percentage of ciliated (ARL13B and Ac-tub double-positive) cells and all cells were ciliated at a similar level; (d, lower panel) Loss of endogenous Nek8 led to truncated cilia but all the NEK8 variants rescued the ciliary length defect except p.Lys157Gln (K157Q). The cilium length was measured by the length of ARL13B signal in cilia. (e) Loss of Nek8 disrupted the recruitment of ANKS6 into cilia, which was fully rescued by all NEK8 variants, except p.Lys33Met (K33M) that only showed a partial rescue. (f) The percentage of GPR161-positive cilia were not affected by the expression status of NEK8. (g) The percentage of PC2-positive cilia and the fluorescence intensity of PC2 in cilia were both diminished by Nek8 deficiency, although (h) IB showed that PC2 was present at comparable protein levels. (g) Different NEK8 variants exhibited different ability to rescue the ciliary trafficking of PC2. (i) Primary skin fibroblasts were isolated from healthy (WT) and p.Arg45Trp (Fam 1 and Fam 7, III-3) male donors. These cells were analysed by IF to visualize PC2 in cilia, with lower mean intensity seen for the two mutant cells. The percentage of PC2-positive cilia and the mean intensity of ciliary PC2 were quantified in >100 cilia from each group. Results from three independent experiments were statistically analysed and plotted as mean ± SD. (j) HEK293T cells transfected with Myc-tagged wild type NEK8 (WT) or the p.Arg45Trp variant (R45W) were subjected to immunoprecipitation (IP) with anti-Myc antibody or normal mouse IgG (mIgG). The precipitates were then subjected to Phos-tag SDS-PAGE to separate the phosphorylated NEK8 (arrow) from the non-phosphorylated form and analysed by IB. The relative density of phosphorylated NEK8 was quantified from four independent experiments, statistically analysed, and plotted as mean ± SD. (c-g, i, j) ***, P < 0.001; **, P < 0.01; *, P < 0.05; n.s., no statistically significant differences.

Figure 4.. Elevated DNA damage signaling in renal cell lines expressing NEK8 variants.

a. Representative images of γH2AX signaling in IMCD3 cells with the indicated genotypes. Scale bar is 50 μm. Graph represents the mean of three independent experiments. Error bars represent sd. At least 150 nuclei per experiment were analyzed for >10 γH2AX foci, **P <0.01, *P <0.05. b. Representative images of γH2AX signaling in patient derived tubuloids with the indicated genotypes. Scale bar is 50 γm c. The percentage of γH2AX positive cells from three independent experiments was quantified. Line represents the mean. Error bars are sd. **P <0.01, *P <0.05.