Functional analysis of Na/H exchanger 9 variants identified in patients with autism and epilepsy.

Na/H exchanger isoform 9, NHE9, finely tunes the pH within the endosomal lumen to regulate cargo trafficking and turnover. In patients with autism, genetic approaches have revealed deletions, truncations and missense mutations in the gene encoding NHE9 (). To help establish causality, functional evaluation is needed to distinguish pathogenic mutations from harmless polymorphisms. Here, we evaluated three previously uncharacterized NHE9 variants, P117T, D496N, and Q609K reported in patients with autism and epilepsy. We show that NHE9-DsRed localizes to recycling endosomes in HEK293 cells where it significantly alkalinizes luminal pH, and elevates accumulation of transferrin. All three NHE9 variants were expressed and localized to endosomal compartments, similar to wild-type NHE9. In contrast to previously characterized NHE9 variants, we observed no loss-of-function with respect to endosomal pH homeostasis and transferrin endocytosis. These findings suggest that the three NHE9 substitutions analyzed in our study are either benign polymorphisms or may have a cell-type specific or regulatory function not detected in our cell culture model. Our findings highlight the importance of combining the use of cellular studies of function with sequencing technologies that capture genomic variation in patients.