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Published on: August 24, 2013
Use of patient-derived cell models for characterization of compound heterozygous hypomorphic C2CD3 variants in a
Zachary T Sentell1,2, Lina Mougharbel2, Zachary W Nurcombe1,2
1Department of Human Genetics, McGill University, 3640 rue University, Montreal, QC, H3A 0C7, Canada.
Background:
Primary ciliopathies are a heterogeneous group of rare disorders predominantly caused by autosomal-recessive genetic variants that disrupt non-motile ciliary function. They often manifest as a syndromic phenotype, frequently involving the kidney. Biallelic pathogenic variants in C2CD3 disrupt ciliogenesis and Sonic Hedgehog (SHH) signaling, resulting in a severe ciliopathy (Orofaciodigital syndrome XIV, OMIM 615948). We present compound heterozygous missense variants in C2CD3 that partially disrupt ciliary function in a patient with isolated renal disease.
Methods:
Exome sequencing identified biallelic C2CD3 missense variants (p.Pro168Leu; p.Thr2079Met). Patient-derived fibroblasts and urinary renal epithelial cells (URECs), and human RPE-1 C2CD3 knockout (KO) cell-lines were used for in vitro studies.
Results:
Cilia length was significantly shorter in patient-derived fibroblasts compared to an unaffected sibling (2.309 vs. 2.850 μm, P < 0.0001), while URECs showed significantly shortened cilia (2.068 vs. 2.807 μm, P < 0.0001) and a 40.8% reduction in ciliation (P < 0.001). The latter was not observed in fibroblasts, suggesting a kidney-specific effect. SHH signaling was dysregulated in patient cells as expression of GLI3 activator protein and GLI1 mRNA was significantly reduced. C2CD3 localization to the basal body was significantly reduced in patient URECs. Finally, rescue experiments in C2CD3 KO RPE-1 cells corroborated these findings by demonstrating a reduced capacity to restore ciliogenesis for each variant.
Conclusion:
Biallelic hypomorphic missense variants in C2CD3 may contribute to an isolated nephronophthisis phenotype with impaired ciliogenesis and SHH signaling. Our findings underscore the importance of functional testing to characterize candidate gene-disease relationships in patients with nephropathy of unknown etiology.
Insights
New C2CD3 gene variants cause kidney disease by impairing cilia function and Sonic Hedgehog (SHH) signaling. This study highlights the role of C2CD3 in isolated renal ciliopathies.
Area of Science:
- Genetics
- Cell Biology
- Nephrology
Background:
- Primary ciliopathies are genetic disorders affecting non-motile cilia, often involving kidney disease.
- Pathogenic variants in C2CD3 cause severe ciliopathies like Orofaciodigital syndrome XIV.
- This study investigates novel C2CD3 variants in a patient with isolated kidney disease.
Purpose of the Study:
- To characterize the functional impact of compound heterozygous missense variants in C2CD3.
- To investigate the role of C2CD3 in kidney ciliogenesis and Sonic Hedgehog (SHH) signaling.
- To explore the genetic basis of isolated nephronophthisis.
Main Methods:
- Exome sequencing identified biallelic C2CD3 missense variants.
- In vitro studies utilized patient-derived fibroblasts, urinary renal epithelial cells (URECs), and C2CD3 knockout cell lines.
- Cilia length, ciliation rates, SHH signaling pathway components (GLI3, GLI1), and C2CD3 localization were analyzed.
Main Results:
- Patient cells exhibited significantly shortened cilia and reduced ciliation, particularly in URECs, suggesting a kidney-specific effect.
- SHH signaling was dysregulated, with reduced GLI3 activator and GLI1 mRNA expression.
- Reduced C2CD3 localization to the basal body and impaired ciliogenesis rescue in knockout cells confirmed variant pathogenicity.
Conclusions:
- Biallelic hypomorphic C2CD3 missense variants can cause isolated nephronophthisis.
- Impaired ciliogenesis and SHH signaling are key mechanisms in C2CD3-related kidney disease.
- Functional testing is crucial for diagnosing nephropathy of unknown origin and understanding gene-disease relationships.
