Clinical data for the patient (A). The pedigree of family 1 segregates CM. The arrow points to the proband. (B) The CT scan reveals the absence of the corpus callosum. The red arrow points to the dilated posterior horn of the lateral ventricle, and the red triangle indicates the upwardly enlarged third ventricle. (C) Sanger sequencing confirmed the RAB11A mutation in family members.

The mutations of the RAB11A (NM_004663.5) (A) Lollipop graph shows pathogenic mutations in RAB11A protein reported in the literature and identified in this study. (B) The pie chart shows the distribution of mutations in the exons. The repeatedly reported mutations are marked in red and the mutation of our proband was pointed in red box.

The variant p. Asp124Asn of RAB11A (A,B) The angles among the nearest contact residues and the wild-type or mutated residues were also measured. Wild-type and mutated residues are depicted by orange sticks, while contact atoms are represented by gray colors. Yellow dotted lines indicate hydrogen bonds. (C) Pathogenicity prediction of the RAB11A missense variant (c.370A>C) using multi-algorithm consensus. Functional impact of the RAB11A mutation (p.N124D) was evaluated by bioinformatic tools based on distinct principles. Evolutionary conservation: GERP++, phyloP. Structural/physicochemical properties: SIFT, PolyPhen-2_HDIV/HVAR, FATHMM-MKL. Machine learning integration: Alphamissense, REVEL, CADD, DANN, Eigen. Pathogenicity probability: MutationTaster, LRT, fitCons, GenoCanyon. (D) Multiple-sequence alignment in RAB11A from different species. The variant was pointed out in red. Conserved protein sequence alignment across 7 species validates critical residue position.

Zebrafish genetic manipulation (A) Phylogenetic analysis of RAB11A in Zebrafish, Human, and Other Species. The phylogenetic tree was constructed using amino acid sequences of RAB11A homologs, generated with MEGA 12 via the Neighbor-Joining method (Bootstrap = 1000 replicates) and visualized using iTOL. Branch node values indicate Bootstrap support rates (shown if ≥ 50%). Scale bar represents amino acid substitutions per site. (B) Amino Acid Sequence Alignment of Key Functional Domains in RAB11A Homologs Across Species (C) Schematic of the rab11a gene (ENSDARG00000041450) and the red sequences represent sgRNA2, 3, 6. The bule part of exon 3 represents the sgRNA knockout target site. (D) The Sanger sequence of mutant rab11a edited by sgRNA2, 3, 6. (E) The procedure of the establishment of rab11a deficient zebrafish model.

Rab11a deficient induces phenotypic anomalies in zebrafish larvae (A) Representative images of zebrafish larvae expressing HuC:eGFP. Brain fluorescence pattern at 5 dpf is displayed (dorsal view). Left, control injected with cas9; right, rab11a crispant. The white dashed line shows the outline of the CNS, which is divided into three parts: forebrain, midbrain, and hindbrain. (B–D) Measurements of brain fluorescence area in control vs rab11a crispant. (B) comparison of brain area data distribution between Cas9 control group and rab11a crispant group; (C) comparison of forebrain area data and frequency distribution between Cas9 control group and rab11a crispant group; (D) comparison of midbrain data between Cas9 control group and rab11a crispant group. (E) comparison of hindbrain data between Cas9 control group and rab11a crispant group. (F,G) Representative local field potential recording showing spontaneous epileptiform events in rab11a crispants. Data were normalized to the mean values of the control group. Error bars indicate standard deviation. Data are shown as mean ± S.E.M. *P < 0.05, **P < 0.01.