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case_images: Biallelic C2CD3 missense variants identified in an individual with isolated renal disease. (A) Pedigree of the index family suggesting autosomal-recessive inheritance in a patient with kidney failure and suspected nephronophthisis. Exome sequencing and phenotype-driven analysis identified C2CD3 (NM_001286577.2) candidate compound heterozygous variants in the proband. (B) Biallelic pathogenic variants in C2CD3 are reported to cause severe ciliopathy disorders (oral-facial-digital syndrome XIV), which are associated with a spectrum of phenotypes, affecting multiple organ systems, including the kidneys. Created with BioRender.com. (C) (I) Exon structure of C2CD3; ATG, start codon; TGA, stop codon. (II) Protein structure of human C2CD3, showing predicted C2 domains. Reported pathogenic variants in OFD cases are represented above by arrow heads. (III) Putative disease-causing missense variants in the proband; Clustal alignment of amino acid sequences of C2CD3 to demonstrate evolutionary conservation from mammalia to ascidiacea for both amino acid residues.
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case_images: There was no family history of renal disease and there was no evidence of consanguinity (Supplemental Figs S1 and S2). Tables S2 and S3). To date, biallelic loss-of-function variants in C2CD3 were reported in 14 humans with a severe syndromic ciliopathy-related disorder (Orofaciodigital syndrome XIV, OMIM 615948), with no reported cases of isolated renal disease (Fig. 1c; Supplemental Table S1). Both C2CD3 missense variants identified in our patient affect highly conserved amino acid residues (Fig. 1c) predicted to be deleterious to protein function by several in-silico models (Table 1). Sanger sequencing of the parents confirmed the variants to be in trans, while none of the healthy siblings carry both variants (Fig. 1a). C2CD3 localizes to the mother centriole, facilitating distal- and sub-distal appendage assembly, essential for basal body maturation and anchoring to the plasma membrane (Fig. 1b).
case_images: Patient-derived cells demonstrate renal-specific ciliogenesis defects. (A) Cilia were significantly shortened in patient fibroblast cells when compared to a healthy control (2.309 vs. 2.850 mum, Mann-Whitney test, P < 0.0001). Data pooled from three-independent experiments. (B) The rate of ciliation between the healthy sibling (74.43%) and the proband (78.28%) was not significantly different (Fisher's exact test; P = 0.0605). (C) Representative cilia images (labelled by acetylated- -tubulin) of fibroblast cells from a healthy sibling (left) and proband (right). (D) Cilia were significantly shortened in patient urine-derived renal epithelial cells (URECs) when compared to a healthy control (2.068 vs. 2.807 mum, Mann-Whitney test, P< 0.0001). (E) URECs had a 40.8% decrease in the proportion of ciliated cells in the proband (16.68%) compared to a healthy control (28.19%) (Fisher's exact test; P < 0.001). (F) Representative cilia images (labelled by acetylated- -tubulin) of URECs from a healthy control (left) and proband (right). (G) Western blot representation of full-length GLI3 (GLI3-FL) protein (activator) expression in proband fibroblast and URECs compared to healthy controls. (G) Western blot showing full-length GLI3 protein (activator) expression in fibroblast and URECs from probands and healthy controls. (H) GLI3-FL protein expression was significantly reduced in proband fibroblasts (relative expression fold-change of 0.1936) and URECs (relative expression fold-change of 0.2701) compared to healthy controls (unpaired two-tailed t-test, P < 0.0001). Data summarized from three independent biological replicates. (I) GLI1 mRNA expression (normalized to GAPDH) was significantly reduced in proband URECs compared to healthy controls (relative expression fold-change of 0.555, unpaired t-test, P < 0.05). Data summarized from three independent experiments. Scale bars represent 10 mum. Data are presented as mean +- standard deviation (SD).
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case_images: There was no family history of renal disease and there was no evidence of consanguinity (Supplemental Figs S1 and S2). Patient dermal fibroblasts had significantly shortened cilia compared to the healthy sibling (P < 0.0001), while the proportion of ciliated cells was not significantly different (Fig. 2a-c). Notably, these patient-derived URECs displayed a more pronounced ciliary defect, not only showing significantly shorter cilia (P < 0.0001), but a 40% decrease in ciliation (P < 0.001) (Fig. 2d-f). Consequently, the downstream transcriptional target of the SHH signaling pathway, GLI1, had significantly reduced expression in the patient URECs compared to healthy controls (Fig. 2i).
case_images: Patient C2CD3 missense variants disrupt C2CD3 localization to the basal body. (A) C2CD3 intracellular localization to the basal body is disrupted in patient URECs compared to healthy controls. C2CD3 co-localization to the basal body was evaluated in serum starved URECs using confocal immunofluorescence microscopy. The Pearson's correlation coefficient (PCC) for C2CD3 to basal body (gamma-tubulin) co-localization was significantly reduced in proband URECs (average PCC = 0.7351) compared to a healthy control (average PCC = 0.7959) (two independent experiments; Mann-Whitney test, P = 0.0046). (B) The Manders overlap coefficient (MOC) for the C2CD3 to basal body (gamma-tubulin) co-localization was also significantly lower in proband URECs (average MOC = 0.8244) compared to a healthy control (average MOC = 0.8753) (two independent experiments; unpaired t-test, two-tailed test, P < 0.0001). (C) Representative images of serum starved URECs from the proband and a healthy control, stained with DAPI, anti-C2CD3, and anti-gamma-tubulin to visualize the intracellular colocalization of C2CD3 to the basal body. Images captured with the ZEISS LSM 880 confocal microscope at 63X/1.4 oil objective. Arrowheads indicate the region of interest at the basal body.
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case_images: Our results indicated a reduction in the colocalization of C2CD3 at the basal body in proband cells, suggesting altered localization dynamics (Fig. 3a-c).
case_images: C2CD3 KO cells expressing patient-specific missense variants recapitulate ciliary defects. (A) C2CD3 colocalization to the basal body (gamma-tubulin) in RPE-1 C2CD3 KO cells expressing GFP-tagged C2CD3 for wild-type (WT) and each patient missense variant (P168L, T2079M), stained with DAPI, anti-GFP, and anti-gamma-tubulin to visualize the intracellular colocalization of GFP-tagged C2CD3 to the basal body. (B) PCC for the colocalization of GFP-tagged C2CD3 to the basal body (gamma-tubulin) revealed no significant differences between the wild-type C2CD3 (PCC = 0.660) and each patient missense variant (p.Pro168Leu PCC = 0.6331; p.Thr2079Met PCC = 0.6293) (one-way ANOVA). MOC for the colocalization of GFP-tagged C2CD3 to the basal body revealed no significant differences between the wild-type C2CD3 (MOC = 0.8368) and each patient missense variant (p.Pro168Leu MOC = 0.8505; p.Thr2079Met MOC = 0.8422) (one-way ANOVA). The proportion of GFP-positive cells was used to estimate transfection efficiency and was not significantly different between conditions (Fisher's exact test; P value = 0.7398). (C) Cilia in serum-starved C2CD3 KO RPE-1 cells expressing GFP-tagged C2CD3 for wild-type (WT) and each patient missense variant (P168L, T2079M) were stained with DAPI, anti-ARL13B to visualize the primary cilium. Normal RPE-1 cells (left) are ciliated and form cilia 3.317 muM in length. (D) C2CD3 KO RPE-1 cells demonstrate a severe reduction in cilia length (2.535 muM); rescue with wild-type C2CD3-GFP restored cilia length (3.427 muM). The P168L C2CD3 protein produced significantly shortened cilia (3.080 muM) compared to the wild-type allele (Kruskal-Wallis ANOVA test, P = 0.0327); the T2079M C2CD3 protein also produced significantly shortened cilia (3.111 muM) compared to wild-type (Kruskal-Wallis ANOVA test, P = 0.0437). (E) C2CD3 LoF RPE-1 cells demonstrate a severe loss of cilia (0.176 mean ciliation). Rescue with wild-type C2CD3-GFP restored ciliation in C2CD3 LoF cells (0.471 mean ciliation). The P168L and T2079M C2CD3 proteins only partially restored ciliation (0.329 and 0.303 mean ciliation, respectively) (Fisher's exact test, P < 0.0001). Data points were pooled from two (N = 2) independent experiments. Images captured with the ZEISS LSM 880 confocal microscope at 63X/1.4 oil objective. Data represent the mean +- standard deviation (SD).
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case_images: We did not detect any significant difference in colocalization of C2CD3 to the basal body in these overexpression models (Fig. 4a and b) for either variant (p.Pro168Leu, p.Thr2079Met), however, both variants displayed a significantly reduced capacity to induce ciliogenesis compared to wild-type, as demonstrated by decreased cilia length and ciliation (Fig. 4d and e). The transfection efficiency of all constructs was comparable (Fig. 4b). These results strongly suggest that the patient variants impair ciliogenesis by reducing both cilia length and ciliation.
case_images: C2CD3 interacts with renal ciliopathy proteins that are essential for basal body anchoring and ciliogenesis. (A) STRING analysis of C2CD3 protein interaction network. Functional enrichment analysis was performed for the gene ontology (GO) cellular compartment, Reactome pathway, and disease associations. (B) (Top) Diagram of the role of C2CD3 in basal body maturation and ciliogenesis. Ciliogenesis can occur through either the intracellular pathway (e.g. fibroblasts, RPE-1 cells) or the extracellular pathway (e.g. polarized renal epithelial cells). C2CD3 is recruited by centriolar protein CEP120 to the distal end of the newly formed centriole during S phase. During procentriole elongation, C2CD3 recruits distal appendage proteins (DAP) and subdistal appendage proteins (sDAP) to the mother centriole. The DAPs mediate the removal of CCP110 from the mother centriole to allow ciliary vesicle docking. C2CD3 is essential for the formation of the transition zone (TZ) and the recruitment of anterograde intraflagellar transport (IFT-B complex) proteins (IFT88 and IFT52) particles which mediate ciliary transport and axoneme microtubule assembly. SHH signaling is initiated at the primary cilium, where it regulates the processing of the GLI3 protein into either a transcriptional activator or repressor. GLI3 processing is disrupted with loss of C2CD3 function. (Middle) Summarized results from patient-derived cells showing mild and renal-specific ciliary defects for each of the described roles of C2CD3. (Bottom) The hypothesized genotype-phenotype relationship for C2CD3-related ciliopathies. Complete loss-of-function variants or severe protein-disrupting variants lead to global ciliary defects that disrupt organ developmental processes, leading to a multi-organ congenital ciliopathy. Hypomorphic variants that cause mild and renal-specific disruption to C2CD3 function may lead to a chronic and degenerative kidney phenotype (nephronophthisis) due to mild loss of ciliary function in the renal tubular epithelium. Created with BioRender.com.
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case_images: C2CD3 protein interaction network revealed its association with proteins crucial for basal body anchoring and ciliogenesis (Fig. 5a, Supplemental Table S5).