Loss of nephrocystin-3 function can cause embryonic lethality, Meckel-Gruber-like syndrome, situs inversus, and renal-hepatic-pancreatic dysplasia

Abstract

Many genetic diseases have been linked to the dysfunction of primary cilia, which occur nearly ubiquitously in the body and act as solitary cellular mechanosensory organelles. The list of clinical manifestations and affected tissues in cilia-related disorders (ciliopathies) such as nephronophthisis is broad and has been attributed to the wide expression pattern of ciliary proteins. However, little is known about the molecular mechanisms leading to this dramatic diversity of phenotypes. We recently reported hypomorphic NPHP3 mutations in children and young adults with isolated nephronophthisis and associated hepatic fibrosis or tapetoretinal degeneration. Here, we chose a combinatorial approach in mice and humans to define the phenotypic spectrum of NPHP3/Nphp3 mutations and the role of the nephrocystin-3 protein. We demonstrate that the pcy mutation generates a hypomorphic Nphp3 allele that is responsible for the cystic kidney disease phenotype, whereas complete loss of Nphp3 function results in situs inversus, congenital heart defects, and embryonic lethality in mice. In humans, we show that NPHP3 mutations can cause a broad clinical spectrum of early embryonic patterning defects comprising situs inversus, polydactyly, central nervous system malformations, structural heart defects, preauricular fistulas, and a wide range of congenital anomalies of the kidney and urinary tract (CAKUT). On the functional level, we show that nephrocystin-3 directly interacts with inversin and can inhibit like inversin canonical Wnt signaling, whereas nephrocystin-3 deficiency leads in Xenopus laevis to typical planar cell polarity defects, suggesting a role in the control of canonical and noncanonical (planar cell polarity) Wnt signaling.

Figure 1

Pathology Findings in Patients with Loss-of-Function NPHP3 Mutations (A–D) Autopsy findings in the second affected patient of family 888. (A) Multicystic dysplastic kidneys. The smaller right kidney had a dilated pelvis due to ureteric atresia in utero. The left kidney had a patent, but thinned ureter and reduced urinary output. (B) Higher-magnification histology (×20) shows a picture with renal cysts of variable size and location as well as interstitial fibrosis consistent with multicystic-dysplastic kidney disease. (C) Congenital pancreatic disease with smaller and larger cysts and fibrosis. Pancreatic acini have normal appearance (×40). (D) Liver histology reveals ductal plate malformation with hyperplastic biliary ducts and congenital hepatic fibrosis (×40). (E) Low-magnification histology of a renal cross-section (×2.5) from the first affected patient of family 806 that is consistent with multicystic dysplastic kidney disease. (F) Renal biopsy specimen of the proposita of family 960 displaying diffuse glomerulocystic kidney disease (×40). Please note glomerular structures within the cysts.

Figure 2

NPHP3 Mutations in Four Families The upper panel shows the pedigree structure of the families, and the lower panel shows sequence chromatograms illustrating the predicted NPHP3 loss-of-function mutations in the described families. Mutations segregated with the disease status in each family (P indicates paternally inherited allele; M indicates maternally inherited allele). In case of a homozygous mutation, a heterozygous electropherogram of one of the parents is shown for reasons of clarity. (A) Sequence chromatogram depicting the deletion of the canonical acceptor splice site nucleotides at position −1_2 of the intron 19 splice acceptor site predicted to result in out-of-frame transcripts and a premature stop. (B) On the left, a sequence chromatograph of the affected individual of family 917 showing a heterozygous G → A exchange which predicts an p.Arg973Gln amino acid substitution is displayed. On the right, a heterozygous C → T exchange predicting a stop codon (p.Gln1114X) is shown. (C) Sequence chromatograph showing a heterozygous C → T exchange that predicts a stop codon (p.Arg577X). (D) Sequence chromatogram illustrating the heterozygous G → A exchange at position +5 of the intron 13 splice donor site resulting in skipping of exon 13 and an out-of-frame transcript that leads to premature termination of translation (Figure S2).

Figure 3

Cystic Kidney Disease in Compound-Heterozygous Nphp3^pcy/ko Mice and Situs Inversus Totalis in Nphp3^ko/ko Embryos Compound-heterozygous Nphp3^pcy/ko mice (A–C) develop cystic kidney disease, whereas control (data not shown) and monoallelic Nphp3^pcy/ko (D–F) mice did not exhibit an obvious renal phenotype. Hematoxylin-eosin stainings of a cryosectioned kidney from a Nphp3^pcy/ko mouse (A) and a Nphp3^pcy/wt mouse (D) are shown. Immunofluorescence staining with antibodies against acetylated α-tubulin (green) as a cilia marker (nuclei, blue) of kidney cryosections from Nphp3^pcy/ko mice (B) demonstrates that epithelial cells of renal cysts carry monocilia that are longer than renal monocilia from control mice (E). (C) and (F) show higher magnifications of the images shown in (B) and (E). Homozygous Nphp3^ko/ko embryos (G–J) have situs inversus and die during embryonic development. Situs inversus in Nphp3^ko/ko embryos is indicated by left-sided tail flexion (G) and mirror-image arrangement of the heart (H). Histological examination of Nphp3^ko/ko embryos confirms dextrocardia (I) and position of the stomach on the right side (J), consistent with situs inversus totalis. Normal situs arrangement was noted in heterozygous Nphp3^ko/wt and control embryos (K–N).

Figure 4

Interaction of Nephrocystin-3 and Inversin (A–C) V5-tagged inversin was coexpressed with N-terminally FLAG-tagged full-length nephrocystin-3 (Flag.NPHP3 FL) or FLAG-tagged CD2AP protein (Flag.CD2AP) as a negative control. Expression of V5-tagged inversin in cellular lysates was confirmed by immunoblotting with V5 antibodies (A). After immunoprecipitation with FLAG antibody, coprecipitating V5-tagged inversin was detected with V5-specific antiserum (B). As control, FLAG-specific antibodies detected precipitated Flag.Inversin and Flag.CD2AP (C). (D–F) In the reverse experiment, V5-tagged full-length nephrocystin-3 (V.5NPHP3 FL) was coexpressed with Flag-tagged inversin or FLAG-tagged CD2AP. The additional approximately 60 kDa band visible in all lanes (B, C, E, and F) represents the heavy chain. The 100 kDa bands that are visible in (E) are nonspecific bands that also appeared upon long exposure time in (B).

Figure 5

Inversin and Nephrocystin-3 Inhibit Dishevelled-1-Induced Canonical Wnt-Signaling Activity (A) Both inversin and NPHP3 inhibit Dvl1-induced activation of a TCF/LEF-1-dependent luciferase reporter construct (TOPFlash) in HEK293T cells. A combined expression of both NPHP proteins leads to a further suppression of the luciferase activity. A luciferase reporter construct with mutated TCF/LEF-1 binding sites (FOPFlash) showed no significant background stimulation. At least four independent experiments were done, each in triplicate. Dishevelled-induced stimulation was always above 20-fold over background. Data was normalized to β-galactosidase expression. p values were calculated with the two-tailed Student's t test. (B–E) Morphogenetic cell movements during gastrulation and neurulation are defective upon NPHP3 knockdown in Xenopus laevis embryos and suggest a role in the PCP Wnt pathway. Gastrulation movements are delayed after NPHP3 knockdown by Morpholino-antisense oligonucleotide injected into two dorsal blastomeres (B). A clone of cells was labeled in the 64-cell stage with fluorescent dextrane, and the cell movements were followed by fluorescent microscopy at stage 12. Upper and lower panels show bright-field and fluorescent images of identical embryos at gastrulation. Incomplete closure of neural folds (C) and phenotypes of shortened body axis and dorsal bending (D) were observed at later stages, suggestive of disrupted PCP/Wnt signaling. This phenotype was dose-dependent and could be partially rescued by coinjection of NPHP3-RNA (E). The phenotypic changes were scored with the convergent extension index (CEI).