Recessive nephrocerebellar syndrome on the Galloway-Mowat syndrome spectrum is caused by homozygous protein-truncating mutations of WDR73

Galloway-Mowat syndrome (GMS) is a neurodevelopmental disorder characterized by microcephaly, cerebellar hypoplasia, nephrosis, and profound intellectual disability. Jinks et al. extend the GMS spectrum by identifying a novel nephrocerebellar syndrome with selective striatal cholinergic interneuron loss and complete lateral geniculate nucleus delamination, caused by a frameshift mutation in WDR73.

Here we associate a novel protein-truncating mutation in WDR73 with a nephrocerebellar syndrome on the Galloway-Mowat syndrome spectrum (herein referred to as NCS) characterized by abnormal cerebral cortical growth without polymicrogyria or heterotopia, cerebellar hypoplasia with granule layer aplasia, aberrant visual pathway development, depletion of striatal cholinergic interneurons, and focal segmental glomerulosclerosis.
Our studies of the WDR73 interactome demonstrate that WDR73 interacts with proteins vital to cell cycle and survival, including -(TUBA1B), b-(TUBB4B), and -(TUBG1) tubulin, HSP-70 (HSPA1A/HSPA1B), HSP-90 (HSP90AA1), p70S6 kinase (RPS6KB1), and CAD (CAD; carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase), a mTORC1-regulated multienzyme complex that mediates de novo pyrimidine synthesis (Ben-Sahra et al., 2013;Robitaille et al., 2013). In vitro, recombinant pathogenic WDR73 variants have increased interactions withand b-tubulin and HSP-90. Fibroblasts from affected children have a cell cycle disruption that substantially slows division, alters the interphase microtubule network and cell morphology, and accelerates senescence. The cell cycle defect can be rescued with wildtype WDR73. Our detailed neuropathological analysis differentiates WDR73-associated NCS from other forms of Galloway-Mowat syndrome and extends the clinical pathological spectrum. Our observations together with those of Colin et al. (2014) support a critical role for WDR73 in the proliferation and survival of human brain and kidney cells.

Materials and methods Patients
The study was approved by Institutional Review Boards of Lancaster General Hospital, Boston Children's Hospital, the University of Arizona, the University of Exeter, the DDC Clinic for Special Needs Children, and the Broad Institute. Parents consented on behalf of their children. Thirty patients with NCS (mean age 9.2 years, range 1.4 to 28 years) originated from three different endogamous Old Order Amish demes and hailed from six states: Pennsylvania, Ohio, Indiana, Michigan, Colorado and Montana. A Bulgarian child with NCS [5 years old at time of diagnosis and unrelated to the WDR73 c.766dupC patient described by Colin et al. (2014)] was added to the study through additional screening at Boston Children's Hospital (see below).

Genetic mapping
Genetic mapping was conducted as previously described (Puffenberger et al., 2004). Genotyping was performed using GeneChipÕ Mapping 10K and 50K Assay Kits (Affymetrix) and single nucleotide polymorphism (SNP) data were analysed using Microsoft Excel. Genotype data came from Affymetrix GeneChip Õ Human Mapping 10K Xba 142 and 50K Xba Arrays and SNP positions came from Affymetrix genome annotation files. Genotype data from multiple affected individuals were examined for shared blocks of homozygosity assuming mutation and locus homogeneity. SNP data from 100 healthy Old Order Amish females were used to estimate population-specific allele frequencies. Two-point LOD scores [logarithm (base 10) of odds] were calculated for each SNP using an approach similar to Broman and Weber (1999). For each block of shared homozygous SNPs, we calculated a cumulative two-point LOD score ('location score') (Broman and Weber, 1999) that indicated the relative probability that the disease gene resided in the homozygous block.

Exome sequencing
Exome sequencing was performed at the Broad Institute using the Agilent SureSelect All Exon Kit (v.1, 38 Mb) as previously described (Puffenberger et al., 2012).

Neuropathology
In Case 1, the fixed brain was first examined macroscopically, and then microscopically using multiple paraffin embedded blocks. In Case 2, a limited number of paraffin blocks were received from the NICHD Brain and Tissue Bank for Developmental Disorders at the University of Maryland. Standard histological and immunohistological methods were used as described previously (Harding et al., 2015) with the addition of immunocytochemistry for choline acetyltransferase (Chat22 goat polyclonal antibody 1:100, #AB144P, Millipore).
WDR73 expression in human brain: tissue processing and immunohistochemistry Frontal neocortex and cerebellar specimens were obtained from three control subjects who died of non-neurological causes (mean age 5.8 years; two male, one female; Brain and Tissue Bank for Developmental Disorders, University of Maryland, mean post-mortem interval = 14 h, range 11-16 h). Cortical cytoarchitecture of specimens was intact. Human tissue specimens were either fixed in 4% paraformaldehyde and paraffin embedded or flash frozen at À 70 C. Tissue blocks were microtome sectioned at 7 mm and five representative sections per case were probed with WDR73 antibodies (Novus Biologicals, rabbit polyclonal, 1:200) overnight at 4 C. Sections were then probed with biotinylated secondary antibodies for 1 h at room temperature and visualized using avidin-biotin conjugation (Vectastain ABC Elite; Vector Labs) with 3,3'-diaminobenzidine. Dehydrated sections were mounted with coverslips (Permount). Lightmicroscopy images were acquired using a Leica DM4000 B microscope. All human tissue was obtained using protocols approved by the Temple University Institutional Review Board.

Cell proliferation assay
Dermal fibroblasts seeded at equal densities on 6-or 12-well plates in DMEM with 10% FBS were cultured (medium replaced every 2-3 days) until NHDFs reached confluency ( Fig. 4) or until heterozygous parent cells reached 50-75% confluency ( Supplementary Fig. 1). Cells were then washed with phosphate-buffered saline (PBS; pH 7.2), fixed for 15 min with 4% paraformaldehyde in PBS at 21 C, washed with sterile water, stained with 0.1% crystal violet in 10% ethanol for 20 min, and washed three times with sterile water. Plates were dried and scanned on an Epson flatbed scanner. Cells were then extracted with 10% acetic acid and the absorbance of the extract was measured with a spectrophotometer at 590 nm. Data were analysed using Student's t-test ( = 0.05) with Microsoft Excel.

Immunofluorescence microscopy
Fibroblasts and HEK-293T cells cultured on uncoated German glass coverslips were rinsed with PBS at 37 C and fixed with 4% paraformaldehyde at 21 C for 15 min. Cells were then prepared for immunofluorescence microscopy and documented as described previously (Puffenberger et al., 2012) Supplementary   Fig. 2, cells were fixed in ice-cold 4% paraformaldehyde and probed with WDR73 antibodies (Novus NBP1-82219) overnight at 0 C and then with Texas Red Õ or FITC conjugated anti-rabbit IgG secondary antibody. Coverslips were mounted with Fluoromount-G TM (Electron Microscopy Sciences). To synchronize fibroblasts in mitosis, doublethymidine block was performed as described previously (Robitaille et al., 2013). All experiments were replicated two to five times.
Co-immunoprecipitation assays were performed as described elsewhere (Puffenberger et al., 2012) with the following exceptions: clarified lysates were incubated with the anti-FLAG M2 affinity gel for 2 h at 4 C followed by 3-4 15-min washes in wash buffer with 2.5-5% Tween-20 (Sigma), protease inhibitors, phosphatase inhibitors, and 1 mM PMSF. Immunoprecipitated proteins were eluted from the affinity gel with 3 Â FLAG peptide (150 ng/ml), and immunoblotted as described above.
HEK-293T cells were transfected with FuGENE6 (Promega) according to the manufacturer's protocol and cultured for 40-48 h before immunofluorescence microscopy, immunoblotting, or coimmunoprecipitation. NCS patient fibroblasts were transfected by nucleofection using the Amaxa Õ Human Dermal Fibroblast Nucleofector Õ Kit (Lonza) and a Nucleofector 2b NHDF program optimized for cell viability following nucleofection.

Genetic mapping
Using 10 000-and 50 000-marker SNP microarrays, we genotyped five affected Amish children and identified a shared 8.26 Mb autozygous block on chromosome 15 that was further refined with nine additional NCS patients to a 3.96 Mb autozygous block (flanked by rs2048271 and rs959181) that contained 69 genes (Fig. 1C). A single child from Indiana had a rare recombination event between rs4359399 and rs713468 that narrowed the minimal autozygous region to 700 kb. Exome sequencing of DNA from seven children with NCS identified a novel homozygous single base pair deletion (WDR73 c.888delT) in all affected individuals. We used Sanger sequencing to confirm that the WDR73 c.888delT frameshift was homozygous in all 27 NCS children and heterozygous in parents.
An additional 800 children with idiopathic nephrotic syndrome were screened using microfluidic multiplex PCR (Fluidigm) and next-generation sequencing (Halbritter et al., 2012;Sadowski et al., 2015). A second homozygous frameshift variant, WDR73 c.766dupC (p.Arg 256Profs*18) was identified in a Bulgarian child with NCS. The WDR73 c.766dupC variant was also identified recently in an unrelated child of Turkish descent with NCS (Colin et al., 2014).
The 3.96 Mb Amish autozygous block contained a second truncating variant in WHAMM (c.1264_ 1270delATAAAAG) (NM_001080435.2) on the same haplotype as the WDR73 mutation. Genotype analysis of all affected individuals revealed that the Indiana Amish NCS patient described above (3 years old) was homozygous for only the WDR73 variant, and heterozygous for the WHAMM mutation. Her clinical course was consistent with NCS ( Fig. 1A) although she had not yet developed nephrotic syndrome. As kidney disease onset is variable in NCS/Galloway-Mowat syndrome, this finding was not unusual. Three additional affected siblings, not available for follow-up, were also considered likely to be homozygous for only the WDR73 mutation and wild-type for the WHAMM mutation (genotypes inferred from parents and an unaffected sister). We were unable to identify any clinical differences between these three individuals and those who were doubly homozygous for both WDR73 and WHAMM variants. In our single non-Amish proband from Bulgaria, we excluded WHAMM mutations through Sanger sequencing.
Phenotype of nephrocerebellar syndrome on the Galloway-Mowat spectrum Table 1 summarizes core phenotypic features of 30 NCS patients (27 confirmed and three inferred WDR73 c.888delT homozygotes) ages 1.4 to 28 years (mean age 9.2 years). Irritability, congenital roving nystagmus, and visual impairment were early postnatal signs of NCS. Head growth was slow ( Fig. 1A and B) and psychomotor development was severely delayed in all domains. Affected children did not effectively communicate or use their hands, and only 10% achieved independent ambulation (Table 1). Most children with NCS developed extrapyramidal movements that included a combination of axial dystonia and limb chorea. Severe visual impairment was associated with progressive optic atrophy. The majority of affected children had irregular or reversed sleep-wake patterns.
Multifocal seizures began during infancy or early childhood in 41% of affected children. EEGs were characterized by slow, disorganized background, absent posterior (occipital) rhythm, poor sleep-wake differentiation, multifocal sharp and spike-slow wave discharges from various cortical regions and rarely, high-voltage modified hypsarrhythmia. Serial MRIs revealed diffuse cerebral atrophy, thinning of the corpus callosum, and hypoplasia and progressive atrophy of cerebellar tissue (Fig. 1B).

Neuropathology
Brain tissue was examined post-mortem from two affected females who died from complications of renal failure at ages 3.5 (Case 1) and 9.3 years (Case 2); pathological findings were similar. Both brains were small, with fixed weights of 636 g (normal for age, 1100 g) and 681 g (normal for age, 1250 g), respectively, and most striking were small sclerotic cerebella with prominent folia ( Fig. 2A and B). The hindbrain of Case 1 weighed 50 g, only 7.9% of total brain weight (expected 12%); this was accompanied by ventricular dilatation, a thin corpus callosum, and atrophic optic nerves ( Fig. 2C and D).
The cerebral cortex showed normal lamination with no obvious gliosis or cell death. Pathological changes were found in the visual system, striatum, and cerebellum. Optic nerve atrophy was confirmed microscopically (Fig. 2C and D), and both magnocellular and parvocellular neurons of lateral geniculate nuclei were nearly absent, obliterating the normal six-layer organization ( Fig. 2E and F). No degenerative changes or gliosis were present in the basal ganglia, but the magnocellular cholinergic interneurons in the striatum were small and scarce ( Fig. 2G and H). By comparison, medium spiny neurons appeared normal.
The cerebellum in both affected children was markedly abnormal. Folia of the cerebellar hemispheres and (A) Postnatal brain growth is slow in children with NCS (grey-shaded area = normal head circumference for age, mean AE 2 SD; red circles = NCS head circumference, mean AE SD). Inset: Children with NCS have no distinctive dysmorphic features, but the forehead characteristically recedes, reflecting relative hypoplasia of the cerebral cortex; post-mortem brain weights are only 50-60% of normal (photos used with parental permission). (B) MRI at 1.5 T (left to right: axial T 2 , coronal T 2 , sagittal T 1 ) shows underdeveloped frontal lobes (asterisks), thin corpus callosum (yellow arrowheads), and severe atrophy of the cerebellar hemispheres (red arrows) and vermis (yellow arrows). Comparable images of the same NCS child, taken at 13 months (upper panel) and 32 months (lower panel), reveal progressive degeneration of cerebellar tissue (arrows). (C) Initial SNP genotyping of five children (inset) mapped recessive NCS to an 8.26 Mb autozygous block of DNA on chromosome 15 (blue arrow). After the initial mapping, we identified nine additional patients that allowed us to narrow the shared homozygous interval to 3.96 Mb. Yellow signal indicates the number of contiguous SNPs shared among NCS individuals. For each block of shared homozygous SNPs, purple signal shows the cumulative two-point LOD score ('location score'), a relative probability, based on population allele frequencies, that the disease gene resides in the homozygous block. Subsequent genotyping of all 27 NCS individuals refined the shared locus to 700 kb, which contained the novel WDR73 c.888delT mutation homozygous in all affected children. vermis were short and stubby ( Fig. 2I and J) and granule cells were profoundly depleted (Fig. 2K). Purkinje neuron morphology was relatively spared but these cells were decreased in number and crowded together. There was excessive Bergmann gliosis. The overlying molecular layer was thin and hypercellular, comprised of Bergmann glia, misplaced NeuN-positive granule cells, and rare Purkinje neurons (Fig. 2K). Calbindin and neurofilament immunostaining of Purkinje neurons revealed spherical dendritic swellings covered in fine spikes ('asteroid bodies'), dysmorphic dendritic trees forming 'weeping willow' arrangements, and occasional Purkinje cell bodies covered in somal sprouts suggestive of deafferentation ( Fig. 2M-O). Globose ('torpedo') swellings of proximal Purkinje cell axons suggested ongoing cell degeneration (Fig. 2P).

Expression of WDR73 variants in cells and tissues
The WDR73   the surface of the fifth WD repeat. This suggests that protein-protein interactions are altered by both pathogenic WDR73 variants. In post-mortem brain tissue, wild-type WDR73 immunoreactivity was observed within pyramidal cells of the cerebral cortex and hippocampus ( Supplementary Fig. 1A and B), and within Purkinje and granule cell neurons of the cerebellum ( Supplementary Fig. 1C), where it largely localized to cytoplasm within somatodendritic domains. Western blot analysis revealed full-length WDR73 (42 kDa) in human cerebral cortex and cerebellum as well as fibroblasts, astrocytes, and U87 glioma cells ( Fig. 4A; see also Supplementary Fig. 2).
NHDFs express full-length WDR73 ( Fig. 4A and B; see also Colin et al., 2014) whereas fibroblasts cultured from WDR73 p.Phe296Leufs*26 heterozygotes display two WDR73 immunoreactive bands ($42 kDa and 35 kDa), each of reduced abundance relative to the single band observed in NHDFs (Fig. 4B). We were unable to culture enough NCS proband (p.Phe296Leufs*26) fibroblasts to reliably detect a WDR73 band by immunoblotting, but would predict reduced abundance of both WDR73 p.Phe296Leufs*26 and p.Arg2 56Profs*18 based on instability of the recombinant versions of these proteins ( Fig. 6F and G).

Cell proliferation assay
Fibroblasts from NCS patients grow poorly in primary culture, especially if cultured at low density and beyond 2-3 passages. Over a 2-week period, fibroblasts from an NCS proband (p.Phe296Leufs*26) and her heterozygous father grew to 16% and 35% of NHDF density, respectively (n = 4 replicates) (Fig. 4C). If seeded more densely (5000 cells per well in a 12-well plate) and cultured until NHDFs reached full confluency, NCS proband and heterozygous parent cells grew to 43.5% and 78% of NHDF density, respectively (n = 4) ( Supplementary Fig. 1G).

Abnormal cell cycle in NCS fibroblasts
Fibroblasts from NCS probands (p.Phe296Leufs*26) were substantially larger (up to 280 mm in diameter) than NHDFs and heterozygous parent fibroblasts (Figs 4D-I and 5E-H). Using immunofluorescence microscopy, we always observed NCS proband fibroblasts in interphase; they did not appear to progress through other phases of the cell cycle. Cytoplasmic WDR73 immunoreactivity was weak in these cells and difficult to distinguish from background ( Fig. 5E-H).

WDR73 associates with the mitotic microtubules during cell division
Reduced proliferation and early senescence of NCS proband fibroblasts suggests that WDR73 plays a fundamental role in the cell cycle. Accordingly, we determined the subcellular localization of WDR73 immunoreactivity across the cell cycle in NHDFs (Supplementary Fig. 3; see also Colin et al., 2014) and parent fibroblasts heterozygous for WDR73 p.Phe296Leufs*26 (Fig. 5). There were no substantial differences in WDR73 immunoreactivity between NHDF and heterozygous parent fibroblasts. During interphase, WDR73 immunoreactivity was diffusely cytoplasmic (Fig. 5A). As cells transitioned into metaphase, WDR73 immunoreactivity associated strongly with -tubulin at the spindle poles and spindle microtubules (Fig. 5B). During anaphase, WDR73 localized to spindle poles, kinetochore microtubules, and interpolar microtubules (Fig. 5C). During telophase, WDR73 immunoreactivity transitioned from the spindle poles along the interpolar microtubules to the central spindle microtubules, to concentrate at the midbody microtubules apposing the stem body, where central spindle microtubules arising from opposite poles of the cell overlap (Fig. 5D). These results were consistent across all cells observed in mitosis. As described above, fibroblasts from patients homozygous for WDR73 p.Phe296Leufs*26 could not be observed in any phase of cell cycle outside of interphase ( Fig. 5E-H).
Transient overexpression of WDR73 wild-type C-terminal V5 fusion protein in fibroblasts from a patient homozygous for WDR73 p.Phe296Leufs*26 rescued the cell cycle defect. Transfected cells that presumably had not yet become binucleate were observed in metaphase (Fig.  6A) and telophase (Fig. 6B) within 5 days of transfection. Mitotic microtubules were only observed in transfected NCS patient fibroblasts. To validate our findings, we also overexpressed WDR73 wild-type C-terminal V5 fusion protein in HEK-293T cells. Recombinant WDR73-V5 localized robustly to the cytoplasm during interphase (data not shown), transitioned to the developing spindle poles and astral and spindle microtubules during prometaphase and metaphase ( Fig. 6C and D), and was prominent at spindle poles, interpolar microtubules, and central spindle/midzone microtubules during anaphase (Fig. 6E).
The WDR73:CAD interaction was not dependent upon phosphorylation of CAD (n = 6) (Fig. 7C), and not diminished by changing CAD Ser1859 to alanine, which eliminates its site of phosphorylation by p70 S6 kinase via the mTORC1 pathway (Robitaille et al., 2013) Supplementary Fig. 4D). We further determined that WDR73 interacts with p70 S6 kinase through coimmunoprecipitation of endogenous p70 S6 kinase by FLAG-WDR73 fusion proteins (n = 11 for wild-type, p.Phe296Leufs*26; n = 7 for p.Arg256Profs*18) (Fig. 7D). To probe the influence of WDR73 on mTORC1 signalling, we knocked down endogenous WDR73 in HEK-293FT and mouse neural progenitor cells and determined that phosphorylation of ribosomal protein S6 (a p70 S6 kinase substrate) was reduced without a decrease in S6 protein abundance ( Fig. 7E and Supplementary Fig. 4E).

Discussion
We describe a novel truncating frameshift mutation in WDR73 associated with a severe nephrocerebellar syndrome (NCS). NCS fits within the broad and evolving phenotypic spectrum of Galloway-Mowat syndrome (OMIM 251300), which is likely genetically heterogenous in aetiology (Pezzella et al., 2010;Keith et al., 2011;Ekstrand et al., 2012;Colin et al., 2014). Our neuropathological findings of delamination of the lateral geniculate nucleus, selective loss of cholinergic interneurons in the striatum and the absence of polymicrogyria and heterotopia in NCS patients are novel, and extend the pathological spectrum of Galloway-Mowat syndrome.
We provide the first description of the WDR73 interactome, as well as evidence, corroborating a recent report by Colin et al. (2014), that WDR73 interacts with mitotic microtubules and is critical for normal cell cycle progression, proliferation and survival of human fibroblasts, podocytes and neuronal subtypes. The two mutations associated with NCS we describe delete the sixth WDR73 C-terminal WD repeat domain and significantly alter the fifth, leading to loss of function, protein instability, and enhanced interactions withand b-tubulin. In fibroblasts, homozygous loss of WDR73 function results in decreased cell proliferation and abnormal cell cycle progression that can be rescued with wild-type WDR73 (Fig. 6). Whether the increase in apoptosis observed by Colin et al. (2014) in patient fibroblasts homozygous for WDR73 p.Tyr43* is a direct effect of WDR73 loss of function or a downstream effect of aborted cell cycle in bi-and multi-nucleated cells remains to be determined. Indeed, occasional binucleate cells were observed in post-mortem NCS cerebella ( Supplementary  Fig. 1).
Localization of wild-type WDR73 to the mitotic spindle, where it interacts with tubulin subunits, raises important questions about how WDR73 influences the function of the spindle and centrosome. Most genes linked to decreased brain size encode centrosomal proteins (e.g. WDR62) (Nicholas et al., 2010;Yu et al., 2010;Chen et al., 2014) and dysfunction of the mitotic spindle is a well-established cause of microcephaly (Gilmore and Walsh, 2013). Abnormal nuclear morphologies observed in NCS proband fibroblasts are consistent with cytokinesis failure, indicating failure of the mitotic spindle (Normand and King, 2010). Overstabilization of interactions between pathogenic Overexpression of WDR73 C-terminal V5 fusion protein (WDR73-V5) in HEK-293T cells. Anti-V5 immunofluorescence is in green; anti-tubulin in red. During pro-metaphase (C) and metaphase (D) recombinant WDR73-V5 colocalizes with -tubulin at the mitotic spindle and aster microtubules. (E) WDR73-V5 localizes to the spindle poles, the kinetochore microtubules and the midzone microtubules during anaphase. Scale bars: A-E = 10 mm. (F-G) Western blots of recombinant N-terminal FLAG WDR73 fusion proteins (F) and WDR73 C-terminal V5 fusion proteins (G) overexpressed for 40-48 h in HEK-293T cells. Anti-COX IV was labelled as a protein loading control. The abundance of FLAG-WDR73 p.Phe296Leufs*26 (F296Lfs*26) was 2.6-to 5.6-fold lower and FLAG-WDR73 p.Arg256Profs*18 (R256Pfs*18) was 1.6-to 6.5-fold lower than that of FLAG-WDR73 wild-type (wt) across four replicates despite transfection of equivalent amounts of plasmid DNA, suggesting instability of the truncated proteins.
WDR73 fragments and tubulins may hinder normal movement of WDR73 along mitotic microtubules, slow spindle microtuble dynamics (Colin et al., 2014), and/or slow the large amount of protein and vesicular trafficking along mitotic microtubules during cell cycle (Hu et al., 2012;Agromayor and Martin-Serrano, 2013;Green et al., 2013).
Slow proliferation of NCS fibroblasts might also reflect dysfunction of the mTORC1 signalling pathway. Our data suggest that WDR73 interacts in a complex with the mTORC1-regulated p70 S6 kinase and its natural substrate, CAD (Ben-Sahra et al., 2013;Robitaille et al., 2013). CAD is responsible for de novo pyrimidine synthesis during S-phase (Ben-Sahra et al., 2013;Robitaille et al., 2013). Knockdown of Wdr73 in mouse neural progenitor cells resulted in decreased phosphorylation of ribosomal protein S6 (the p70 S6 kinase substrate) ( Fig. 7 and Supplementary Fig. 4). Moreover, inactivation of mTORC1 signalling in Rptor knockout mice results in (38 kDa) overexpressed in HEK-293T cells co-immunoprecipitated (IP) substantially more endogenousand b-tubulin and HSP-70 than FLAG-WDR73 wild-type (45 kDa). (B) FLAG-WDR73 wild-type, F296Lfs*26, and R256Pfs*18 each co-immunoprecipitated -tubulin at an abundance roughly proportional to abundance of the respective WDR73 construct. (C and D) Co-immunoprecipitation of (C) endogenous CAD, phospho-Ser 1859 CAD, HSP-90, and (D) p70 S6 kinase from lysates of HEK-293T cells overexpressing the WDR73 constructs indicated. (E) Lentiviral transduction of shRNA plasmid constructs A and B targeting WDR73 in HEK-293FT cells successfully knocked down WDR73 and produced a concomitant decrease in ribosomal protein S6 phosphorylation. GAPDH was labelled as a protein loading control. This immunoblot was labelled with the same anti-WDR73 antibody (Novus) used for immunoblotting throughout the paper (validating the specificity of the antibody). microcephaly associated with reduced neural progenitor cell proliferation, slowing of neural progenitor cell cycle, and increased neuroblast apoptosis, as well as proteinuria and progressive glomerulosclerosis (Godel et al., 2011;Cloetta et al., 2013). We attempted to rescue reduced proliferation of NCS fibroblasts with uridine (pyrimidine) supplementation, but did not observe improvements in cell proliferation or morphology (data not shown).
Key functions of WDR73 in vivo remain to be defined, but the consistent and severe NCS phenotype reported here and by Colin et al. (2014) indicates that WDR73 has specific and indispensable roles in generation, proliferation, and/or viability of cortical neuroblasts, development of the cerebellar granule cell layer, survival of cholinergic striatal interneurons, central visual pathway development, and podocyte biology.
Severe cerebellar pathology in NCS most closely resembles classical descriptions of granular layer aplasia (Smeyne and Goldowitz, 1989;Harding and Copp, 2008). In foetal mice, a transient external granular layer arises by proliferation of cells lining the fourth ventricle (Miale and Sidman, 1961). These cells migrate over the external cerebellar surface and continue to proliferate rapidly until a few weeks after birth. The murine external granular layer disappears during the third postnatal week, as its cells migrate inward past Purkinje neurons to form a compact layer of small granule cell neurons, the internal granular layer (ten Donkelaar et al., 2003). Weaver mice (Kcnj6 mutation) have selective and profound depletion of cerebellar granule cells caused mainly by accelerated cellular senescence (Smeyne and Goldowitz, 1989;Harkins and Fox, 2002;Takahashi et al., 2006).
In human cerebellum, intensive cell proliferation occurs within the external granular layer between the 28th and 34th gestational weeks and is still active by the fifth postnatal month (Abraham et al., 2001). The mature number of granule cells will exceed 50 billion, accounting for 75% of neurons in the human brain. Hypoplasia and degeneration of cerebellar tissue observed in NCS reflects massive aplasia and/or depletion of this cerebellar granule cell population and its associated neuropil due to reduced proliferation, accelerated demise, or both (Harding and Copp, 2008). This appears to entrain reactive remodelling and loss of Purkinje neurons, which are markedly dysmorphic, with 'weeping' dendritic branches, spiked dendritic asteroid bodies, somal sprouts, and bulbous 'torpedo' swellings of proximal axons. The presence of somal sprouts on the surface of Purkinje cells was a remarkable finding in both NCS brains. These protrusions are only rarely observed in granule layer aplasia, and have only been reported in Menkes' disease (OMIM 309400), an X-linked disorder of copper metabolism (Harding, 2013). Although deep cerebellar nuclei remain intact in NCS brain, the histopathology suggests a profound disruption of cerebellar feedback to the nervous system, which must contribute significantly to visual (Voogd et al., 2012;Saglam et al., 2014), sensorimotor (Volpe, 2009;Manto and Oulad Ben Taib, 2013), and cognitive (Barton, 2012;Hoche et al., 2014) disabilities of affected individuals.
Selective depletion of striatal cholinergic interneurons alters signalling through cortico-basal-thalamic circuits to produce an array of extrapyramidal movements (i.e. dystonia, chorea, restlessness) (Pisani et al., 2007;Aosaki et al., 2010;Goldberg et al., 2012). We found further evidence of selective neuronal vulnerability within lateral geniculate nuclei, which were depleted of magno-and parvocellular neurons and devoid of their hexalaminar organization. We have no way to determine if this reflects a paucity of anterograde trophic signalling from the retinae to the lateral geniculate nuclei, or if primary neuronal hypoplasia within the lateral geniculate nuclei or striate cortex leads to transneuronal, retrograde atrophy of retinal ganglion cell axons within the optic tracts. Selective injury to any neuronal population within the central visual system appears to entrain degeneration of all neural elements within the pathway (You et al., 2012;Hendrickson et al., 2013).
Glomerular findings in NCS are those of classic steroid non-responsive focal segmental glomerulosclerosis (D'Agati et al., 2011;D'Agati, 2012). Podocyte injury and depletion is common to all forms of focal segmental glomerulosclerosis (Barisoni, 2012;D'Agati, 2012) and was evident on electron micrographs from NCS kidney, which showed effacement and microvillus remodelling of 80-90% of podocytes. Morphological and functional changes of NCS kidney reflect an important but as yet poorly understood role of WDR73 in podocyte biology (Colin et al., 2014). We observed 'striped fibrosis' and tubular atrophy resembling that caused by calcineurin inhibitors, cyclosporine and tacrolimus (Shihab, 1996;Liptak and Ivanyi, 2006). These changes could be non-specific, but may also be a clue to underlying signalling mechanisms relevant to NCS. For example, the mammalian target of rapamycin (mTOR), a key determinant of human cortical growth and organization (Orlova and Crino, 2010;Cloetta et al., 2013;Lim and Crino, 2013;Tsai et al., 2014), also influences podocyte shape and function (Godel et al., 2011;Cina et al., 2012;Jeruschke et al., 2013;Muller-Krebs et al., 2013;Zaza et al., 2014). Recent evidence suggests an important interaction between mTOR and calcineurin pathways in renal cell lines (Basu et al., 2011(Basu et al., , 2012, and this could provide new insights into the pathophysiology of NCS. Although genetic evidence for pathogenicity of WDR73 c.888delT is definitive, the linked truncating variant in WHAMM (c.1264_1270delATAAAAG) (NM_00108043 5.2) merits additional investigation. As the phenotypical outcome of loss of WHAMM function alone is currently unknown we cannot exclude the possibility that patients doubly homozygous for WHAMM and WDR73 mutations may have a modified phenotype. Further long-term clinical assessments of these individuals, or the identification of subjects who are homozygous for WHAMM c.1264_1270 delATAAAAG but heterozygous (or wild-type) for WDR73, may help to clarify this question. However, our data, corroborated by recent observations of Colin et al. (2014), support the assertion that WDR73 loss-of-function is sufficient to cause NCS on the Galloway-Mowat spectrum.