Fig. 1: Rare mutations in APAF1, CASP9, and CASP3 contribute to human neural tube defects.

a Illustration of APAF1, CASP9, CASP3 protein structure with non-synonymous variants identified in 352 NTD cohorts (red arrow) and 224 control group (green arrow). Asterisk indicates the recurrent mutations that affect more than 2 cohorts. b Representative results of sequence alignment including CASP9 R180C, CASP3 Q217H, and APAF1 P335R in different species and caspase subfamily members. Mus, house mouse. Gallus, chicken. Xenopus, clawed frog. Danio, Zebra fish. Drosophila, fruit fly. c Rare mutations in CASP9, especially R180C totally blocked procaspase-9 cleavage in transfected 293T cells, as a result, cleaved-CASP3 was reduced. (Left). CASP3 Q217H impairs spontaneous cleavage of CASP3, as well as downstream PARP. The phosphorylation of Akt at Thr308 site and release of p37 subunit from CASP9 were reduced by Q217H when compared with wild type CASP3 (Right). GAPDH served as a loading control. S, short exposure; L, long time exposure. d Volcano plot of control vs WT or mutant CASP9. Significant differences were detected in R180C samples compared to wild type and Y251C samples (Left). The log2 fold change between the treatment means is plotted on the horizontal axis. The −log10 FDR (adjusted p-values by Fisher’s test) is plotted on the vertical axis. Black points are not statistically significant, and red points are significant at p < 0.01 and fold-change>2. The representative differential expressed gene GH1 identified by RNA-seq was confirmed in the NE-4C cell line by qPCR (Right)