Structural modelling of ZC3H11A gene mutations.

(A) Illustration of exons and mutation sites of ZC3H11A; exons 5-8 of ZC3H11A encode the zf-CCCH_3 protein domain. (B) A structural model of the full-length human ZC3H11A protein was generated using PyMOL; the locations of the four mutation sites are marked. (C) The p.G43E mutation is located in exon 6, while the p.V138I and p.P154L mutations are in exon 8, and the p.S747T mutation is in exon 20. (D) Multiple sequence alignment of the proteins where ZC3H11A mutations are found in multiple species. (E) These mutations can result in a higher degree of conformational flexibility at the corresponding sites, potentially destabilizing the structural domain.

The clinical features and mutations of affected patients

Comparison of ocular biometrics and body weights of Zc3h11a Het-KO (n=14) and WT (n=10) littermates during weeks 4-10 of development.

(A, B) Refraction and axial length in Zc3h11a Het-KO mice. (C) Vitreous chamber depth. (D-F) Anterior chamber depth, lens depth and body weight. The effect of genotype on time-dependent refractive development was assessed through independent samples t-tests. P-values are indicated as follows: *P<0.05, **P<0.01, ***P<0.001 and ****<0.0001.

Electroretinography and immunohistochemical localization of Zc3h11a, PKCα, Opsin-1 and Rhodopsin in WT and Het-KO mice eyes.

(A) Representative scotopic ERG responses from Het-KO and WT eyes at dark 0.01, 3.0 and 10.0 cds/m2. (B, C) Statistical analysis of a-wave and b-wave amplitudes. Mean ± standard deviation, n=12/group. (D-F) Immunofluorescence-stained samples to detect Zc3h11a, bipolar cells, cone and rod cells. (G, H) Lower levels of Zc3h11a and PKCα (E. H) expression were observed in Het-KO mice (I, J) The expression levels of Opsin-1 and Rhodopsin between Het-KO and WT mice. Statistical significance was defined as *P<0.05, **P<0.01 and ***P<0.001, as determined by independent samples t-tests.

TEM showing the retinal ultrastructure of WT and Het-KO mice.

(A, B) Cellular morphology of the INL in which bipolar cells are located in WT (A) and Het-KO mice (B). (C, D) Cellular morphology of the ONL in which the optic cells are located in WT (C) and Het-KO mice (D). (E, F) MB structure in WT mice. (G, H) MB structure of Het-KO mice.

RNA-Seq analysis of molecular and pathway differences between WT control and Het-KO retinas.

(A, B) Volcano maps were utilized to visualize the differentially expressed genes (DEGs) between WT control and Het-KO mouse retinas. (C, D) GO enrichment analysis of DEGs in the Het-KO group (E) KEGG pathway enrichment analysis of DEGs in the Het-KO group.

Effect of Zc3h11a on the expression of genes and proteins related to the PI3K-AKT and NF-κB signalling pathways in the retina.

(A-C) The ZC3H11A, PI3K and AKT gene levels (n=3) were evaluated. (D, E) The IκBα and NF-κB gene levels (n=3). (F) Protein expression levels of ZC3H11A, PI3K, AKT, p-AKT and GAPDH were detected by western blot. (G-I) Quantitative analysis of the ZC3H11A and PI3K levels were normalized to GAPDH, while p-AKT levels were normalized to AKT (n=3). (J) The protein expression levels of IκBα, NF-κB and GAPDH were determined by western blot analysis. (K, L) Quantitative analyses of IκBα and NF-κB were normalized to GAPDH (n=3).

Effect of Zc3h11a on the expression of TGF-β1, MMP-2 and IL-6 in the retina and sclera.

(A, B) Examination of TGF-β1, MMP-2 and IL-6 gene expression in the sclera (A) and retina (B) (n=3). (C) TEM structure of sclera. (D) Determination of TGF-β1 and MMP-2 protein expression levels, normalized to GAPDH, through western blot analysis (n=3).

Schematic representation of the PI3K-AKT-NF-κB signalling pathway in retina.

Generation of Zc3h11a KO mice.

(A) Generation of Zc3h11a KO mice in C57BL/6J background using CRISPR/Cas9 technology. (B-C) Primers for genotyping and examples of genotyping results of Zc3h11a Het-KO mice and wild-type mice

Fundus photographs and HE staining of Het and WT mice at 8th week.

(A. B) Fundus photographs of WT (A) and Het-KO mice (B) (n=3). (C. D) HE staining of retina in WT (C) and Het-KO mice (D) (n=3).

Sequence of oligonucleotides