Base editing-derived models of human WDR34 and WDR60 disease alleles replicate retrograde intraflagellar transport (IFT) and hedgehog signaling defects
Base editing derived cell models shed light on the role of ciliary dynein complex components for intraflagellar transport, hedgehog signaling and differential regulation of genes associated with Golgi intracellular transport using human disease alleles.
Abstract
Cytoplasmic Dynein-2 / IFT-dynein is the only known retrograde motor for intraflagellar transport. Dysfunction of the two intermediate dynein chains WDR34 and WDR60 causes Short Rib Thoracic Dystrophy (SRTD), a human skeletal chondrodysplasias with high lethality. However, individual protein functions are incompletely understood as complete loss of function of WDR34 or WDR60 is lethal in vertebrates and individuals with SRTD carry at least one putative hypomorphic missense allele. Gene knockout is therefore not suitable to study the effect of these human missense disease alleles. Therefore, using CRISPR single base editors, we recreate three different human disease missense alleles, including a novel WDR60 variant, p.Ala968Val identified in this study. Consistent with previous findings in the dynein-2 full loss of function models and patient fibroblasts, mutant cell lines show hedgehog signaling defects as well as disturbed retrograde IFT. Transcriptome analyses reveal differentially regulated expression of genes associated with various biological processes, including regulation of the actin cytoskeleton. Further, we observe differential regulation of genes associated with Golgi intracellular transport. In addition to providing cellular model systems enabling investigations of the effect of human SRTD disease alleles, our findings indicate non-ciliary functions for WDR34 and WDR60 in addition to the established roles as components of the retrograde IFT motor complex in cilia. Base editing derived cell models shed light on the role of ciliary dynein complex components for intraflagellar transport, hedgehog signaling and differential regulation of genes associated with Golgi intracellular transport using human disease alleles.
Does a homozygous splicing variant in WDR78 lead to male infertility with multiple morphological abnormalities of the sperm flagella (MMAF)?
A homozygous WDR78 splicing variant disrupts WD40 domains through aberrant splicing and is associated with MMAF and defective sperm flagellar assembly.
Multiple morphological abnormalities of the sperm flagella (MMAF) represent a well-defined genetic cause of male infertility, combining asthenozoospermia with severe structural defects of the flagellum. To date, more than fifty genes, mainly encoding axonemal or peri-axonemal proteins, have been implicated. Many of these proteins are essential for dynein arm assembly and flagellar motility. WDR78 encodes a WD-repeat protein involved in inner dynein arm formation and ciliary motility in animal models. However, its contribution to human male infertility has not yet been established
A consanguineous North African family with five infertile brothers and a homogeneous MMAF phenotype was investigated. The study combined whole-exome sequencing, in silico variant interpretation and functional validation using a minigene splicing assay.
One affected brother underwent detailed semen analysis according to WHO recommendations. Whole-exome sequencing was performed following a negative result using an extended infertility gene panel. The candidate splice variant was analysed with multiple in silico splicing prediction tools. Its functional impact was assessed using a minigene assay in HEK293T cells, followed by RT–PCR, capillary fragment analysis and Sanger sequencing to characterise the resulting transcripts.
Whole-exome sequencing identified an homozygous splice-site variant in WDR78 (NM_024763.5:c.2113-2A>G). This variant showed an extremely low allelic frequency in population databases (6.9 × 10−7 in gnomAD) and was absent from the 1000 Genomes Project, supporting its rarity and compatibility with an autosomal recessive mode of inheritance. In silico analyses consistently predicted a severe splicing defect. No other variant susceptible to be responsible of the phenotype was identified. Functional assessment using a minigene assay demonstrated activation of a cryptic splice site within exon 15, resulting predominantly in a single aberrant transcript characterised by a 53-bp deletion. This abnormal transcript introduced a frameshift and a premature stop codon, predicting a truncated WDR78 protein lacking the final 99 amino acids, including two WD40 repeats. These domains are essential for β-propeller formation and for protein–protein interactions required for inner dynein arm assembly. Disruption of these structural features provides a coherent molecular explanation for the severe abnormalities of sperm flagellar structure and motility observed in the affected brothers, all of whom presented a homogeneous MMAF phenotype.
This report is restricted to a single consanguineous family, and additional independent cases are needed to confirm the gene–disease association. Functional validation relied on a heterologous cell system, which may not fully reproduce germ cell–specific splicing regulation and protein function.
These findings identifies WDR78 as a novel gene responsible for MMAF and male infertility, justify its inclusion in diagnostic gene panels and underscore the contribution of functional assays to the interpretation of splice-site variants detected by exome sequencing.
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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.
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