De novo mutations (DNMs) validation of selected seven DNVs in trios affected with anencephaly. Chromatograms of the three individuals from one trio (from top to bottom: fetus, father, and mother). (A) BRF1 de novo variant. (B) CNOT3 de novo variant. (C) DMXL2 de novo variant. (D) ORC3 de novo variant. (E) SATB2 de novo variant. (F) SHPRH de novo variant. (G) WIPI1 de novo variant.

Abnormal expression of wipi1 affected zebrafish embryo convergent extension. (A) Zebrafish embryos injected with wipi1-MO (antisense morpholino) show defects of convergent extension, including shortening of the trunk, and crooked or bent tails. To assess the extension defects quantitatively, the phenotypic classes were scored as grade 1 (normal), grade 2 (mild), grade 3 (moderate), and grade 4 (severe) in zebrafish embryos at 24 and 48 h post-fertilization (hpf) (scale bar = 500 μm). (B) Zebrafish embryos injected with wipi1-MO exhibit dosage-dependent convergent extension deficits at 24 and 48 hpf. Injection with wipi1-MO at dose of 2–6 ng showed a significant difference compared with control injection. (C) Overexpression of Wipi1 in zebrafish embryos at 24 and 48 hpf. Injection 200 pg mRNA and 300 pg mRNA resulted in a significant increase of malformed zebrafish. There was no correlation between mRNA dosage and severity of malformation (^∗P < 0.05).

wipi1 mRNA variants affected the convergent extension in zebrafish embryos treated with wipi1-MO. (A)WIPI1 WT mRNA of 100–300 pg rescued the convergent extension defects induced by co-injection of wipi1-MO in zebrafish embryos at 24 and 48 hpf. There was no difference in malformation between embryos co-treated with 100 pg mRNA and wipi1-MO dosage and control embryos untreated with MO or mRNA. (B)WIPI1 mRNA co-injected with wipi1-MO rescued the convergent extension. However, WIPI1 R328Q, G313R, T418M, or L406P mRNA injection failed to rescue the phenotype, while WIPI1 WT mRNA injection compensated for the phenotype (^∗P < 0.05).

WIPI1 variants and their expression of mRNA and protein. Human HEK 293 cells were cotransfected with pIRES2-dsRed and WIPI1 variants. At 48 h post-transfection, the cells were harvested for real-time PCR and Western blotting. (A)WIPI1 mRNA expression level was not influenced by WIPI1 R328Q, G313R, T418M, or L406P mutations. (B)WIPI1 L406P mutation affected WIPI1 protein expression, while WIPI1 R328Q, G313R, or T418M had no adverse effects on WIPI1 protein expression. (C) Relative WIPI1 mutant protein expression analysis. The data were derived from three independent experiments and expressed as mean ± SD. Data for WIPI1 mRNA and expression were normalized by GAPDH for each sample. Error bars represent standard error/deviation of the mean. The asterisk indicates a statistically significant difference between wild-type and L406P mutation (^∗∗P < 0.01; ^∗P < 0.05).