It is concluded that PC2C331S may perturb protein stability and/or polycystin complex formation prior to ciliary/EV trafficking and a C. elegans pipeline for mechanistic classification of conserved ADPKD-associated missense variants is established.
Abstract
Interpreting the pathogenic significance of missense variants in human disease gene candidates remains a major challenge in precision medicine. Autosomal dominant polycystic kidney disease (ADPKD), a common cause of kidney failure, results from mutations in PKD1 or PKD2, encoding polycystin-1 and polycystin-2. Here, we establish C. elegans as a platform for the functional classification of PC2 variants by characterizing PKD-2C180S, the C. elegans ortholog of the likely pathogenic human variant PC2C331S. Using CRISPR/Cas9 genome editing with dual-color fluorescent reporters and super-resolution imaging, we show that PKD-2C180S reduces cell-body protein levels, abolishes ciliary and extracellular vesicle (EV) localization, and eliminates sensory function, phenocopying a pkd-2 null allele. In heterozygous animals, PKD-2C180S is recessive and exerts no dominant-negative effect on wild-type PKD-2 trafficking, protein levels, or function, establishing that PKD-2 is haplosufficient in this model. PKD-2C180S also abolishes ciliary and EV localization of the PC1 homolog LOV-1 and reduces LOV-1 cell-body levels, phenocopying pkd-2 null animals, consistent with PC2 acting as a molecular chaperone for PC1 stability and trafficking. Genetic epistasis analysis shows that PKD-2C180S protein levels and ciliary trafficking defects are intrinsic to the mutant protein and independent of lov-1 and the ciliary kinesin-3 klp-6. Quantitative analysis reveals that LOV-1•PKD-2 is more abundant at the ciliary membrane and more efficiently packaged into EVs than PKD-2 alone in lov-1 mutants. We conclude that PC2C331S may perturb protein stability and/or polycystin complex formation prior to ciliary/EV trafficking. This work establishes a C. elegans pipeline for mechanistic classification of conserved ADPKD-associated missense variants.
In ADPKD renal function is corrupted by the accumulation and growth of fluid-filled cysts. Disruption of the PKD1 gene product, polycystin-1, is the most frequent cause of ADPKD, but the mechanisms that predispose PKD1 to recurrent somatic mutation remain poorly understood. Because current evidence for sequence- and structure-dependent mutational susceptibility is strongest at the human PKD1 locus, we will focus here on mechanisms that may promote somatic PKD1 inactivation. Experimental evidence for polycistin-1 inactivation supports a two-hit pathway, with the first hit being an inherited germline pathogenic mutation in PKD1 and the second hit mutation arising later in a somatic cell to inactivate the gene or lower the gene's dosage to lift a barrier to cyst initiation. An affected kidney can have thousands of cysts, each of which is a clonal lineage arising from an independent mutational second-hit event. Why human PKD1 is prone to inactivation and why the rodent orthologs escape similar mutagenesis is a mystery that, once solved, promises to provide important insights into the molecular mechanisms governing cyst initiation. A step toward that goal came from the characterization of the guanine-rich sequence architecture of human PKD1 that distinguishes it from rodent Pkd1. These guanine-rich tracts are intrinsically susceptible to oxidative damage and can adopt non-duplex secondary structures such as guanine-quadruplex DNA. Although guanine quadruplexes serve important regulatory functions, both oxidized guanine lesions and guanine quadruplex structures can interfere with faithful DNA replication and repair, thereby increasing mutation risk. Here, we discuss PKD1 mutagenesis in the context of the renal inflammatory microenvironment, integrating established principles of sequence-dependent mutagenesis and guanine-rich DNA structure biology with mechanisms of cyst initiation in ADPKD. This synthesis supports a conceptual model in which intrinsic sequence-dependent mutational susceptibility at the human PKD1 locus may interact with localized inflammatory microenvironments characterized by oxidative stress and epithelial proliferation to contribute to recurrent somatic second-hit formation.
L. Hanakahi, Tara Subrahmanyan, Gregory B Vanden Heuvel et al.· BMC Nephrology· 0 citations
Autosomal dominant polycystic kidney disease (ADPKD) accounts for 5-10% of prevalent end-stage kidney failure (ESKD). ADPKD cysts result from a loss of sufficient functional expression of PKD1/Polycystin-1 (PC1) in approximately 80% of families. Kidney disease severity correlates with the extent to which PC1 dosage is reduced below a critical level, and evidence suggests therapeutic benefit from increasing PC1 expression in these conditions. Upstream open reading frame (uORF) translation can reduce translation of a protein's coding sequence. Ribosome profiling data and bioinformatic predictions suggested the presence of conserved PKD1 uORFs, so we sought to explore their biological role. We generated luciferase reporters and two humanized PKD1 5'UTR mouse models with or without single nucleotide edits removing uORF start codons ("delta-uORF") to define active uORFs and test their impact on PC1 translation. PKD1 uORF start codons can robustly initiate translation and delta-uORF conveys a 2-4-fold increase in PC1 protein expression and resultant prevention of kidney cysts in Dnajb11 as well as in Pkd1 missense models. PKD1 uORF1-blocking steric antisense oligonucleotides (ASOs) substantially increase PC1 expression in vitro. PKD1 uORFs play an important role in the low basal expression of wild-type PKD1, and their inhibition represents an opportunity to therapeutically increase PC1 translation in polycystic kidney and liver disease resulting from reduced dosage of PC1.
Zhigui Li, Zi Guo, Soyoung Cho et al.· Journal of Clinical Investig...· 0 citations
The first functional characterization of the cardiomyopathy-associated SMYD1 N101S variant identified in a child with severe infantile cardiomyopathy is provided, establishing a mechanistic link between SMYD1 dysfunction and infantile cardiomyopathy and highlighting the importance of integrating genomic and functional approaches in rare cardiovascular disease.
Marta W. Szulik, Clint Gwynn, Magnus Creed et al.· bioRxiv· 0 citations
Summary Autosomal dominant polycystic kidney disease (ADPKD) arises from mutations in polycystin-1/PKD1 or polycystin-2/PKD2 that induce injury-repair pathways and dysregulate cAMP and Yes-associated protein (YAP) signaling to promote cyst growth. Cystic kidneys with elevated cAMP levels also develop in mouse embryos lacking the BicC family RNA-binding protein 1 (Bicc1). To prevent embryonic lethality, we flanked Bicc1 exon 4 with loxP sites for conditional knockout (cKO) in renal tubules using inducible Pax8-rtTA-driven Cre in adults, or distal segment-specific deletion in utero using Ksp-Cre. Whereas Bicc1-deficient adult kidneys formed relatively few cysts within six months, distal nephron-specific deletion induced aggressive PKD-like disease. Automated renal tubule and cyst quantification (ARTCyQ), a novel machine-learning pipeline to analyze immunofluorescent stainings, revealed increasing ambiguity in nephron segment identity and activation of ADPKD-related pathways, including YAP signaling. Accordingly, transcriptional profiling revealed overlap with PKD and injury-repair signatures, supporting a role for Bicc1 in polycystin signaling.
Céline Gagnieux, F. Bernet, Lona J Kroese et al.· iScience· 0 citations
Summary Congenital heart disease (CHD) remains a major cause of pediatric morbidity and mortality, yet many patients lack a genetic diagnosis. Two studies identified rare deletions in ARHGAP10 (GAP10), a Rho GTPase-activating protein at 4q31.2 in individuals with heterotaxy and atrial septal defects, highlighting GAP10 as a new candidate CHD gene; however, the function of GAP10 has not been investigated. We demonstrate that gap10 deletion disrupts body axis extension, left-right organizer (LRO) formation, and ciliogenesis, leading to severe cardiac looping defects that closely mirror human CHD phenotypes in Xenopus. Gap10 localizes to basal bodies of motile cilia in multiciliated cells, where it regulates basal body and apical F-actin organization by recruiting focal adhesion kinase (FAK) to specialized ciliary-adhesion complexes. Our findings implicate GAP10 as a clinically relevant, genetically supported, and functionally validated regulator of CHD and ciliogenesis, underscoring the power of functional genomics for discovering rare disease genes.
Ewelina Sochacka, Savanna Hinson, David R. Shook et al.· iScience· 0 citations
Plasma proteomic analysis of patients with CILD40 identified significant enrichment of pathways related to platelet activation, complement and coagulation cascades and differential signatures in CILD40, highlighting the potential of plasma proteomics for understanding distinct pathogenic mechanisms across PCD subtypes.
Siming Kong, Mingshuo Wang, Xuedong Wang et al.· Phenomics· 0 citations