Figure 5

Identification of PAK2 mutations in human fetuses with NTDs. A) Two nonsynonymous (c.451C>T, p.P151S; c.758A>C, p.E253A) and one recurrent splice site mutation (c.289‐3T>A) in the PAK2 gene are identified among five patients with NTDs and validated using Sanger sequencing. B) Sequence conservation analysis of the amino acid sequences at P151 and E253 of PAK2 among multiple vertebrates. C1–C2) The relative levels of PAK2 and key components in BMP signaling in brain tissues of health controls and human NTDs carrying PAK2 mutations as shown using the NanoString nCounter RNA assay. C1, Color bar represents log2 fold‐change; C2, the relative levels of each gene are presented as the mean ± SEM. D) The levels of PAK2 (F = 7.612, p = 0.0052; p < 0.01 for WT‐PAK2 versus PAK2‐P151S) in N2a cells transfected with WT‐PAK2 and PAK2‐P151S. E) The levels of PAK2 (F (2, 15) = 26.41, p < 0.0001; p = 0.0002 for WT‐PAK2 versus PAK2‐E253A) in N2a cells transfected with WT‐PAK2 and PAK2‐E253A. F) The levels of PAK2 in N2a cells transfected with PAK2‐P151S (p = 0.298 for MG132; p = 0.016 for BAF A1) or PAK2‐E253A (p = 0.019 for MG132, p = 0.049 for BAF A1) were treated with MG132 or Baf‐A1 for 6 h. G) The levels of ADP in N2a cells transfected with WT‐PAK2, PAK2‐P151S and PAK2‐E253A. p = 0.007 for WT‐PAK2 versus PAK2‐P151S, p < 0.0001 for WT‐PAK2 versus PAK2‐E253A. H) The levels of pPAK2 (Ser141) in N2a cells transfected with WT‐PAK2, PAK2‐P151S and PAK2‐E253A. p = 0.026 for WT‐PAK2 versus PAK2‐P151S, p = 0.036 for WT‐PAK2 versus PAK2‐E253A. One‐way ANOVA with Dunnett's multiple comparison test (D–H). n = 6 cultures for each group.