ANKS 6 is a central component of a nephronophthisis module linking NEK 8 to INVS and NPHP 3

Nephronophthisis (NPH) is an autosomal recessive cystic kidney disease that leads to renal failure in childhood or adolescence. Most NPHP gene products form molecular networks. We have identified ANKS6 as a new NPHP family member that connects NEK8 (NPHP9) to INVERSIN (INVS, NPHP2) and NPHP3 to form a distinct NPHP module. ANKS6 localizes to the proximal cilium and knockdown experiments in zebrafish and Xenopus confirmed a role in renal development. Genetic screening identified six families with ANKS6 mutations and NPH, including severe cardiovascular abnormalities, liver fibrosis and situs inversus. The oxygen sensor HIF1AN (FIH) hydroxylates ANKS6 and INVS, while knockdown of Hif1an in Xenopus resembled the loss of other NPHP proteins. HIF1AN altered the composition of the ANKS6/ INVS/NPHP3 module. Network analyses, uncovering additional putative NPHP-associated genes, placed ANKS6 at the center of the NPHP module, explaining the overlapping disease manifestation caused by mutations of either ANKS6, NEK8, INVS or NPHP3. NPH is the most frequent genetic cause of renal failure in children, presenting with cystic kidney disease combined with extrarenal manifestations such as retinitis pigmentosa (Senior-Løken syndrome), liver fibrosis, cerebellar vermis hypoplasia (Joubert-syndrome), situs inversus or cardiac malformations 1,2. Since most NPHP gene products localize to the cilium or its appendages, NPH, the related Joubert-syndrome and Meckel-Gruber syndrome (MKS) have been termed ciliopathies 3. Although more than a dozen causative genes have been identified, a surprisingly large proportion of patients with NPH (approximately 60%) do not have a mutation in any of the known NPHP genes 4. Most NPHPs display domain architectures typical for adaptor molecules involved in protein-protein interactions, and form large protein networks 5,6. Hence, a remaining challenge is the identification of the missing components to understand how these protein complexes exert their developmental and tissue-specific functions. Although NPHP members engage in multiple protein-protein interactions, four distinct sub-networks have been identified, the NPHP1-4-8, the NPHP5-6, the NPHP2-3-9 and the MKS modules 5,6,7. However, how specific complexes are assembled and how the composition of individual complexes is regulated, is currently unknown. NEK8, a NimA (Never in mitosis A)-related serine-threonine kinase is mutated in NPHP9. INVS recruits NEK8 and NPHP3 to the cilium and has only been shown to interact with NEK8 directly 7,8,9. To obtain insight in the molecular function of NEK8 in NPH, we expressed NEK8 in human embryonic kidney (HEK293T) cells and identified interacting proteins by mass spectrometry (MS) 10. This approach identified ANKS6, a protein containing nine N-terminal ankyrin repeats and a C-terminal sterile alpha motif (SAM), as a potential binding partner (Supplementary Table 1); co-immunoprecipitation assays confirmed the interaction between NEK8 and ANKS6 (Supplementary Fig. 1). An Arg823Trp missense mutation of Anks6 (SamCystin, Pkdr1) has recently been identified as the underlying cause of cystic kidney disease in the Han:SPRD +/Cy rat 11. Anks6 was Hoff et al. Page 2 Nat Genet. Author manuscript; available in PMC 2014 February 01. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript detected at the proximal segment of the cilium in murine inner medullary collecting duct (IMCD) cells (Fig. 1a and Supplementary Fig. 1), similar to the localization of INVS, NPHP3 and NEK8 at this compartment 7,12. To analyze the role of Anks6 during embryogenesis, we used morpholino antisense oligonucleotide (MO)-mediated depletion in zebrafish. Injection of two independent anks6 MOs caused pronephric cyst formation (Fig. 1b, c and Supplementary Fig. 2). The cystic phenotype caused by anks6 depletion in the pronephric tubule was identical to nek8 and nphp3 morphants 13,14 (Fig. 1d, e) and combined knockdowns had an additive effect on cyst formation (Supplementary Fig. 2). In addition, laterality defects, detected by cmlc2 staining of early heart looping were observed in anks6 depleted zebrafish, and were comparable to nphp3and nek8-deficient embryos (Fig. 1f, g). Since unilateral injections allow a tissue restricted knockdown and analysis of organ specific phenotypes, we turned to the Xenopus model to analyze the developmental events in renal formation in further detail. Both nek8 and anks6 were expressed during Xenopus development, and were enriched within the proximal Xenopus pronephros at later developmental stages (Supplementary Fig. 3). Bilateral knockdown of anks6 by MO (Supplementary Fig. 4) resulted in gross body edema typical for a renal excretory defect (Fig. 2a) 15,16, also observed after nphp3 (Supplementary Fig. 5) and invs depletion 17. Depletion of either nek8 or anks6 resulted in a striking simplification of the proximal pronephros convolute (Fig. 2b, c), a phenotype previously also reported for the knockdown of Invs 17. Co-expression of a MO-insensitive mRNA coding for nek8 or anks6 respectively rescued the abnormalities, supporting the specificity of the observed phenotypes (Fig. 2b and Supplementary Fig. 4) The nek8 MO-mediated defects were partially rescued by coexpression of anks6 (Fig. 2c). This suggests that both proteins have common molecular effects, allowing Anks6 to partially substitute for Nek8. Early pronephric progenitor and later segmentation markers were not affected by nek8 or anks6 depletion (Supplementary Fig. 5 and Fig. 2d). The reduction in SGLT-1Kand NKCC2-positive pronephros segments indicated a shortening of the proximal and intermediate tubule (Fig. 2d). These data support the overlapping roles of Nek8 and Anks6 during early tubular morphogenesis, and are consistent with the phenotypic changes after nphp3 (Supplementary Fig. 5) and invs depletion 17. The striking genetic and phenotypic similarities between NEK8 and ANKS6 suggested that ANKS6 might be involved in human cystic kidney disease, presenting with a nephronophthisis-like clinical syndrome. Mutation analysis of our NPHP cohorts yielded eight patients from six families with six different homozygous ANKS6 mutations (Table 1, Supplementary Fig. 6 and 7): two families with truncating mutations (c.2054_2064del, p.His685Profs*12 [B6794], c.2370_2372delTCA, p.Tyr790* [A649]), two families with splice site mutations (c.1973-3C>G [A3114], c.2512-2A>C [NPH316]) and two families with non-synonymous missense mutations (c.934G>C, p.Ala312Pro [A3121], c.1322A>G, p.Gln441Arg [B7397]). All affected individuals had polycystic kidney disease (PKD) with an early (infantile) onset, except for family NPH316 (juvenile). While patients with missense mutations showed normal-sized cystic kidney disease without extrarenal manifestations, splice site and truncating mutations were associated not only with an enlarged renal size, but also with severe extrarenal defects such as hypertrophic obstructive cardiomyopathy, aortic stenosis, pulmonary stenosis, patent ductus arteriosus, situs inversus and periportal liver fibrosis (Supplementary Fig. 6). Only in family NPH316 (Supplementary Fig. 6, Table 1), a splice site mutation (c.2512-2A>C) did not lead to early onset ESRD and liver involvement, possibly due to the localization in intron 14, affecting splicing of the last exon only. Hoff et al. Page 3 Nat Genet. Author manuscript; available in PMC 2014 February 01. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript Structural heart defects are often associated with laterality defects, and can result from defective cardiac looping during embryogenesis, which we observed in Anks6, Nek8 and Nphp3 deficient zebrafish embryos (Fig. 1g, f). INVS and NPHP3 mutations cause ventricular and atrial septal defects, pulmonary and aortic stenosis, and ventricular hypertrophy 18–20. Four of our patients with ANKS6 mutations (A3114, B6794, A649, NPH316) had aortic stenosis, causing obstructive cardiomyopathy in one patient (A649); another one of them (A3114) displayed additional pulmonary stenosis (Table 1), linking these three NPHP proteins by their clinical manifestations. The conserved missense ANKS6 Gln441Arg mutation (B7397) did not alter the interaction with other NPHP proteins nor its ciliary localization (Supplementary Fig. 7), but failed to rescue the renal phenotype in Xenopus, confirming the clinical relevance of this mutation (Supplementary Fig. 6). These observations indicate that ANKS6 is a novel member of the NPHP gene family and part of the phenotypically distinct NEK8-INVS-NPHP3 subgroup, which is characterized by structural heart defects in addition to infantile nephronophthisis and situs inversus. To determine whether ANKS6 is part of a larger NPHP-associated protein network, we evaluated its interaction with other NPHP proteins. The kinase domain and the region between amino acid 259 and 312 of NEK8 was required for the interaction with the ankyrin repeat domain of ANKS6 (Fig. 3a, b and Supplementary Fig. 8), which also recognized NPHP3 and INVS (Fig. 3c), but not the structurally related ankyrin repeat protein Diversin (Supplementary Fig. 8). When NEK8 was precipitated from cells co-expressing INVS, the immobilization of INVS was strongly enhanced by the presence of ANKS6 (Supplementary Fig. 8). Similarly, when NEK8 was co-expressed with NPHP3, ANKS6 connected NEK8 to NPHP3 (Fig. 3d). Analysis of cilia in various tissues and model systems did not detect a role for ANKS6 in cilia formation or length control, but knockdown in Xenopus epidermal cells showed a mild defect of polarized orientation (Supplementary Fig. 9). Juvenile cystic kidney (jck) mice carry a missense mutation in Nek8. Anks6 localization was identical in wild type and jck mice, even detectable in cyst lining cells (Supplementary Fig. 9). Consistent with a role for INVS to recruit other NPHPs, endogenous Anks6 was lost from the proximal segment of cilia