Loss-of-function mutations in WDR73 are responsible for microcephaly and steroid-resistant nephrotic syndrome: Galloway-Mowat syndrome

Abstract

Galloway-Mowat syndrome is a rare autosomal-recessive condition characterized by nephrotic syndrome associated with microcephaly and neurological impairment. Through a combination of autozygosity mapping and whole-exome sequencing, we identified WDR73 as a gene in which mutations cause Galloway-Mowat syndrome in two unrelated families. WDR73 encodes a WD40-repeat-containing protein of unknown function. Here, we show that WDR73 was present in the brain and kidney and was located diffusely in the cytoplasm during interphase but relocalized to spindle poles and astral microtubules during mitosis. Fibroblasts from one affected child and WDR73-depleted podocytes displayed abnormal nuclear morphology, low cell viability, and alterations of the microtubule network. These data suggest that WDR73 plays a crucial role in the maintenance of cell architecture and cell survival. Altogether, WDR73 mutations cause Galloway-Mowat syndrome in a particular subset of individuals presenting with late-onset nephrotic syndrome, postnatal microcephaly, severe intellectual disability, and homogenous brain MRI features. WDR73 is another example of a gene involved in a disease affecting both the kidney glomerulus and the CNS.

Figure 1

Identification of WDR73 Mutations, Gene Structure, and Protein Structure (A) Pedigrees of families A and B. The affected status is indicated by filled symbols, and the allele status is given below each tested individual. Representative chromatograms show the homozygous c.129 T>G (p.Tyr43^∗) and c.766dupC (p.Arg256Profs^∗18) mutations and the wild-type allele. Red arrows indicate the position of the nucleotide changes. Abbreviations are as follows: het, heterozygous; and hom, homozygous. (B) Schematic overview of the eight exons (black boxes) of human WDR73 and of the six WD40 repeats (green hexagons) predicted in WDR73. Asterisks (for the gene) and red diamonds (for the protein) denote the two mutations described in this article. (C) Model of residues 73–370 of human WDR73 (GenBank AAF28942.1). WDR73 is shown as a rainbow-spectrum cartoon showing the six-bladed β sheets. Blades are numbered 1–6, and the direction of the polypeptide chain is indicated by a color ramp (N-terminal in blue and C-terminal in red) along the length of the ribbon.

Figure 2

Neuroimaging and Renal Pathological Analysis (A) MRI features of three affected individuals with GMS. Sagittal T1 (a, d, and e) and T2 (b) images are shown. Microcephaly with no gyration defect, a thin corpus callosum, and cortical and cerebellar (arrows) atrophy without brainstem anomalies were common in all affected individuals. Axial T2 images (c and f) are shown. Ventricular dilations with moderate subtentorial atrophy were observed in individuals II-4 in family A and II-3 in family B. (B) Kidney sections of individuals with WDR73 mutations. Individual II-3 in family A showed (a) a marked collapse of the glomerular tufts associated with podocyte hypertrophy (arrow; trichrome-safranin stain; 63× magnification) and (b) the presence of a FSGS lesion surrounded by a layer of enlarged podocytes. The adjacent capillary loops are retracted, and the capillary lumen is small (arrowhead; methenamine-silver stain; 100× magnification). Individual II-3 from family B showed (c) diffuse podocyte hypertrophy and periglomerular fibrosis and large vacuoles in some cells (arrow; methenamine-silver stain; 63× magnification) and (d) the presence of an FSGS lesion (arrowhead; trichrome-safranin stain in light green; 100× magnification).

Figure 3

WDR73 Localization in Kidneys and Brain (A) Immunofluorescence in normal human kidney sections shows the localization of WDR73 (in green) and synaptopodin (in red). During kidney development, strong labeling of WDR73 was observed from the S-shaped-body stage (white arrow) to the capillary-loop stage (arrowhead) and decreased along glomerulus maturation. (B) Immunoperoxidase staining of endogenous WDR73 in the infant CNS. WDR73 localized to the cell body of Purkinje cells in the cerebellum (left panel, arrows), in pyramidal neurons in the cerebral cortex (middle panel, arrows), and in their projecting axons (left and middle panels, arrowheads). In the white matter (right panel), WDR73 antibodies stained the cell body of astrocytes (arrows) and their cell processes (black arrowheads) and also endothelial cells of cerebral capillaries (white arrowheads). WDR73 was not detected in oligodendrocytes (red arrowheads). Scale bars represent 20 μm.

Figure 4

WDR73 Localization during the Cell Cycle Immunolabeling of WDR73 (in red), γ-tubulin (in cyan), a marker of the centrioles, and α-tubulin (in green) in control fibroblasts. During interphase, a weak cytosolic staining of WDR73 was observed. However, during mitosis (from prophase to anaphase), WDR73 relocalized to microtubule asters and the cleavage furrow. Scale bars represent 10 μm.

Figure 5

Phenotype of WDR73-Depleted Podocytes and Fibroblasts (A) Hoechst staining in fibroblasts (left panel) and podocytes (right panel). A-II-4 fibroblasts and WDR73-depleted podocytes displayed alterations in nuclear morphology, including budding, multilobulation (white arrows), shrinkage, or fragmentation. For generating the graphical quantification of the number of nuclear alterations, 100 nuclei from ten random fields in each condition were observed and classified as having either a normal or an abnormal nuclear shape. The Student’s t test was used for comparing differences between A-II-4 and control fibroblasts. The mean ± SEM is shown (^∗p < 0.05; ^∗∗p < 0.01). Scale bars represent 20 μm. (B) Graph showing the results of the colorimetric cell-proliferation assay (MTT) in fibroblasts. The viability of A-II-4 cells was significantly lower than that of control cells. The Student’s t test was used for comparing differences between A-II-4 and control fibroblasts. The mean ± SEM is shown (^∗p < 0.05; ^∗∗p < 0.01). (C) Immunolabeling of annexin V shows a higher rate of apoptosis in A-II-4 fibroblasts than in control fibroblasts. Scale bars represent 10 μm.

Figure 6

Effect of WDR73 Invalidation on Microtubule Polymerization and Nuclear Shape in Undifferentiated Podocytes Immmunolabeling of α-tubulin (in green) and lamin (in red), a marker of the nuclear envelope, before and after nocodazole treatment. After microtubule depolymerization induced by 4 hr incubation with nocodazole (T0), the shape of the nucleus in WDR73-depleted cells became similar to that in control cells, and microtubule aggregates within the cytosol were observed (white arrows). After microtubule repolymerization (T60), WDR73-depleted cells contained a nonhomogenous repartition of microtubules cables (arrow head) that did not properly diffuse within the cytosol, and the nucleus became multilobulated. A graph at T0 and T60 shows the number of multilobulated cells during the microtubule-regrowth assay. The Student’s t test was used for comparing differences between WDR73-depleted and control podocytes. The mean ± SEM is shown (ns, not significant; ^∗p < 0.05; ^∗∗p < 0.01). Scale bars represent 20 μm.

Figure 7

Effect of WDR73 Depletion on Microtubule Polymerization and Cell Shape in Differentiated Podocytes (A) Immunostaining of WDR73 (in red) and α-tubulin (in green) in differentiated podocytes. WDR73 localized adjacent to some microtubule cables (arrow heads). WDR73 depletion was associated with abnormal morphology and altered WDR73 localization, which was rescued by the wild-type protein. (B) Nocodazole treatment in differentiated podocytes was conducted before immunolabeling with γ-tubulin (in red) and α-tubulin (in green) antibodies. After 5 min of microtubule regrowth, WDR73-depleted podocytes displayed a delay in microtubule polymerization, which was restored by reintroduction of the wild-type protein. Scale bars represent 20 μm.