Base editing derived models of human WDR34 and WDR60 disease alleles replicate retrograde IFT and hedgehog signaling defects and suggest disturbed Golgi protein transport

Abstract

Cytoplasmic Dynein-2 / IFT-dynein is the only known retrograde motor for intraflagellar transport. Dysfunction of WDR34 and WDR60, the two intermediate chains of this complex, causes Short Rib Thoracic Dystrophy (SRTD), human skeletal chondrodysplasias with high lethality. 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. Using CRISPR single base editors, we recreated three different patient missense alleles.Consistent with previous findings in the dynein-2 full loss of function models and patient fibroblasts, mutant cell lines showed hedgehog signaling defects as well as disturbed retrograde IFT. Transcriptomics analyses revealed differentially regulated expression of genes associated with various biological processes, including regulation of the actin cytoskeleton. Further, we observed 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.

Disease-associated mutations in WDR34 lead to diverse impacts on the assembly and function of dynein-2

“…As we were preparing this work for publication, Antony and colleagues (Antony et al, 2022) released a preprint that included analysis of two of the WDR34 mutants, p.R182W and p.G393S, included here using base editing of IMCD3 cells. This more precise approach to engineering mutants clearly has advantages in terms of engineering mutations into the endogenous locus and therefore avoiding limitations of overexpression.…”

“…Their work defines more subtle, and sometimes contrasting phenotypes to ours. For both mutants, no significant changes to the ability of cells to form cilia, or in cilia length (Antony et al, 2022). Both mutants showed an increase in IFT88 at the ciliary tip, consistent with a defect in retrograde IFT.…”