Three‐layered proteomic characterization of a novel ACTN4 mutation reveals its pathogenic potential in FSGS

Genetic diseases constitute the most important cause for end‐stage renal disease in children and adolescents. Mutations in the ACTN4 gene encoding the actin‐binding protein alpha‐actinin‐4, are a rare cause of autosomal dominant familial focal segmental glomerulosclerosis (FSGS). Here, we report the identification of a novel, disease‐causing ACTN4 mutation (p.G195D de novo ) in a sporadic case of childhood FSGS using next generation sequencing. Proteome analysis by quantitative mass spectrometry of patient derived urinary epithelial cells indicated that ACTN4 levels were significantly decreased compared to healthy controls. By resolving the peptide bearing the residue aspartic acid on position 195 (p.195D) we could verify that the mutant protein is less abundantly expressed as compared to the wildtype protein. Further analyses revealed that the decreased stability of p.G195D is associated with increased ubiquitylation in the vicinity of the mutation site. We next defined the ACTN4 interactome which was predominantly composed of cytoskeletal modulators and LIM domain containing proteins. Interestingly, this entire group of proteins, including several highly specific ACTN4 interactors, was globally decreased in the patient derived cell clones. Taken together, these data suggest a mechanistic link between ACTN4 instability and proteome perturbations observed in the ACTN4 interactome. Our findings advance the understanding of dominant effects exerted by ACTN4 mutations in FSGS. This study illustrates the potential of genomics and complementary, high‐resolution proteomics analyses for the assesment of novel, potentially causative genetic variants in the context of rare disorders. Support or Funding Information DFG