Identification of patient mutation. (A) Patient information and clinical diagnosis. (B) The distribution ratio of SNV/InDel sites in different regions of the whole genome. (C) The distribution ratio of SNV/InDel sites of different functional types. (D) The distribution ratio of SNV types of conversion/transversion. (E,F) Sanger sequence of the target sequence.

Establishment of cdh23 gene knockout lines. (A) The schematic diagram of the cdh23 gene target site. The gene is 450.8 kb in length and has 72 exons. Exon 18 is the target for CRISPR/Cas9 gene editing in zebrafish cdh23. (B) Design of cdh23 gene knockout target, the uppercase letter is the exon, the lowercase letter is the intron, “___” is the detection primer, “ ~ ~ ~ ~” is the target site sequence, and “=====” is the PAM sequence. The distance of the two target sites is 115 bp; (C,D) Partial DNA and the corresponding amino acid sequence of cdh23 line 1 and line 2 mutant, respectively. The blue box indicates the missing sequence. Line 1 has a total deletion of 34 bp, and protein translation is terminated prematurely. Line 2 missed 143 bp, and protein translation also terminated prematurely. (E,F) DNA agarose gel of cdh23 line 1 and line 2, respectively.

Knock out of cdh23 resulted in impaired hair cell function. The function of hair cells is evaluated by performing a YO-PRO-1 uptake experiment at 6 dpf. The ear hair cell (A) and lateral neuromast (B) uptake YO-PRO-1, while in the cdh23 homozygous mutant, the ear hair cell (C) and lateral neuromast (D) (arrow) do not have YO-PRO-1 fluorescence signal. (E) Genotyping results confirmed that the embryos with impaired function of hair cells are homozygous mutants. (F) The numbers of hair cells in each group, ***, significant difference, p<0.001.

The results of startle response for zebrafish juveniles. (A) Under the action of prepulse of different intensities, the movement distance of cdh23 homozygous mutants and heterozygous juveniles was significantly lower than that of the wild type at the same time. (B) Under the stimulation of prepulse of different intensities, in cdh23 homozygous mutants and sibling juveniles, the movement speed of sibling juvenile fish is significantly lower than that of wild type. All zebrafish juveniles used developed to 6 dpf. (C) Genotyping results of zebrafish juveniles after startle response.

Transcriptomics landscape with cdh23^−/−. (A) 1240 genes were identified as DEGs (p value < 0.05 and ∣log₂FC∣ > 1) with 324 being up-regulated and 916 being down-regulated. (B) Gene distribution according to expression level between case and control; (C,D) The important terms and pathways are closely related to phenotypic characteristics based on DEGs identified in this study.

Network construction and visualization based on specific pathway and gene. (A,B) Key genes involved in glycolysis/gluconeogenesis and purine metabolism and their interactions; (C,D) The expression heatmap of these genes between the control and the cdh23^−/− group. (E) Genes closely related to cdh23 in the string database. (F) RT-qPCR analysis evaluating the expression change of genes related to cdh23.

ATP compensation partially recovered the cdh23 mutant phenotype. (A) The hair cells in ear and around the eye can uptake YO-PRO-1 dye while the cdh23 mutant embryos cannot (C), (A’): the hair cells in the control inner ear, (C’): the hair cells in cdh23 mutant embryos. The ATP supply of control (B), hair cells were specifically stained by YO-PRO-1 dye, in cdh23 mutant (D), after ATP adjunction, the function of hair cells in inner ear were partially recovered, while the hair cells around the eye still impaired. (A’–D’) is the otic regions of (A–D), respectively. (E) The numbers of hair cells in each group, ns, no significant difference, ***, significant difference, p<0.001.

Schematic diagram of the potential molecular mechanism regarding cdh23 on congenital hearing loss. In WT, candidate genes (clic5b, myof, myo7bb) may be associated with the cytoskeleton of actin and participate in the movement of cilia in inner ear hair cells. Purine and amino acid metabolism yield sufficient ATP, and when ATP binds to the P2X7 receptor, these genes (atp1b2b, cacna1fa) may mediate the opening of related ion channels, thereby completing the acoustic-electric conversion. While in cdh23^−/− embryos, purine metabolism is disrupted, resulting in a shortage of ATP, leading to dysfunction of the ion channel P2X7 receptor, and hence interrupting the acoustic-electric conversion, resulting in congenital hearing loss.