A Deregulated Stress Response Underlies Distinct INF2-Associated Disease Profiles

Significance Statement Inverted formin 2 (INF2) is the key regulator of a stress response?calcium-mediated actin reset, or CaAR?that reorganizes the actin cytoskeleton of mammalian cells in response to calcium influx. INF2 has been linked to the podocytic kidney disease focal segemental glomerulosclerosis (FSGS) and to cases of the neurologic disorder Charcot?Marie?Tooth disease that are accompanied by FSGS. The authors show that >50 disease-associated INF2 variants lead to deregulation of CaAR in cell lines, in Drosophila nephrocytes, and in cells from patient cells with these disorders. Their findings suggest that CaAR can be used as a sensitive assay for INF2 function and for robust evaluation of disease-linked variants of this formin. This work also highlights the use of quantitative cellular assays in assessing effects of disease-associated mutations to better understand complex disease phenotypes. Background Monogenic diseases provide favorable opportunities to elucidate the molecular mechanisms of disease progression and improve medical diagnostics. However, the complex interplay between genetic and environmental factors in disease etiologies makes it difficult to discern the mechanistic links between different alleles of a single locus and their associated pathophysiologies. Inverted formin 2 (INF2), an actin regulator, mediates a stress response?calcium mediated actin reset, or CaAR?that reorganizes the actin cytoskeleton of mammalian cells in response to calcium influx. It has been linked to the podocytic kidney disease focal segemental glomerulosclerosis (FSGS), as well as to cases of the neurologic disorder Charcot?Marie?Tooth disease that are accompanied by nephropathy, mostly FSGS. Methods We used a combination of quantitative live cell imaging and validation in primary patient cells and Drosophila nephrocytes to systematically characterize a large panel of >50 autosomal dominant INF2 mutants that have been reported to cause either FSGS alone or with Charcot?Marie?Tooth disease. Results We found that INF2 mutations lead to deregulated activation of formin and a constitutive stress response in cultured cells, primary patient cells, and Drosophila nephrocytes. We were able to clearly distinguish between INF2 mutations that were linked exclusively to FSGS from those that caused a combination of FSGS and Charcot?Marie?Tooth disease. Furthermore, we were able to identify distinct subsets of INF2 variants that exhibit varying degrees of activation. Conclusions Our results suggest that CaAR can be used as a sensitive assay for INF2 function and for robust evaluation of diseased-linked variants of formin. More broadly, these findings indicate that cellular profiling of disease-associated mutations has potential to contribute substantially to sequence-based phenotype predictions.