Structural and evolutionary analysis of ZC3H11A mutations in high myopia.

(A) Enomic organization of ZC3H11A: Exon-intron structure (exons 1-20) with four identified missense mutations (orange arrows: c.128G>A/p.G43E, c.412G>A/p.V138I, c.461C>T/p.P154L, c.2239T>A/p.S747T). Domain architecture showing the zf-CCCH_3 zinc finger domain (exons 5-8) harboring three mutations (G43E, V138I, P154L). (B) Full-length ZC3H11A structural model: Predicted tertiary structure (PyMOL v2.5) with mutation sites highlighted: G43E (exon 6, orange), V138I/P154L (exon 8, green and blue), S747T (exon 20, purple). (C) Mutation localization: Schematic mapping of mutations to exons: G43E (exon 6), V138I/P154L (exon 8), S747T (exon 20). Exon sizes scaled proportionally. (D) Cross-species conservation: Multiple sequence alignment of ZC3H11A orthologs showing absolute conservation of mutated residues. (E) DynaMut2-predicted conformational flexibility changes: 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

Zc3h11a Het-KO mice exhibit myopic shifts in refractive parameters.

(A) Het-KO mice showed myopia in diopter. (B, C) Axial length and Vitreous chamber depth increased elongation in Het-KO mice at weeks 4 and 6. (D-F) No genotype-dependent differences in anterior chamber depth, lens thickness, or 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.

Zc3h11a Het-KO mice exhibited bipolar cell dysfunction, accompanied by reduced abundance of the key bipolar cell marker protein PKCα.

(A) Representative scotopic ERG responses from Het-KO and WT eyes at dark 0.01(2.02 log cd·s/m2), dark 3.0 (0.48 log cd·s/m2and 10.0 (0.98 log cd·s/m2). (B, C) Quantification of a-wave (photoreceptor function) and b-wave (bipolar cell function) amplitudes. Het-KO mice (n=12) show significant b-wave reduction at dark 3.0 and dark 10.0 vs. WT (n=12). No a-wave differences observed (p>0.1). (D-F) Immunofluorescence-stained samples to detect Zc3h11a, PKCα (key bipolar cell marker protein), Opsin-1 (key cone photoreceptors marker protein) and Rhodopsin (key rod photoreceptors marker protein). (G, H) Zc3h11a and PKCα protein abundance reduced in the retina of Het-KO mice. (I) Western blot analysis showed that the PKCα protein content in the retina of Het-KO mice was decreased. (J) No differences in Opsin-1 or Rhodopsin protein abundance. 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 abnormalities ultrastructure of Zc3h11a Het-KO mice.

(A-B) Inner nuclear layer (INL). (A) WT mice, normal bipolar cell morphology; (B) Zc3h11a Het-KO pathological features include enlarged perinuclear gaps (black arrowheads), cytoplasmic edema (blue arrowheads), thinned/lightened cytoplasm in bipolar cells. (C-D) Outer nuclear layer (ONL). (C) WT mice and (D) Zc3h11a Het-KO mice Het-KO, no significant differences in photoreceptor nucleus morphology. (E-H) Photoreceptor membrane discs (MB). (E-F) WT mice, tightly stacked, uniformly arranged membrane discs. (G-H) Zc3h11a Het-KO structural disruptions include the outer layer falls off, the local distribution is sparse, and the arrangement is chaotic and loose (red arrow).

RNA-Seq reveals pathway dysregulation in the retina of Zc3h11a Het-KO mice.

(A) Volcano plot of differentially expressed genes (DEGs): 769 total (303 upregulated, 466 downregulated in Het-KO vs. WT retinas. (B, C) Gene Ontology (GO) enrichment analysis: Biological Process: Zinc ion transmembrane transport (GO: 0071577, photoreceptor maintenance), Negative regulation of NF-κB signaling (GO: 0043124, scleral remodeling). Molecular Function: Calcium ion binding (GO: 0005509, phototransduction), Zinc ion transmembrane transporter activity (GO: 0005385, retinal zinc homeostasis). (D) KEGG pathway enrichment analysis: Key pathways: PI3K-AKT signaling, MAPK signaling.

Dysregulation of PI3K-AKT and NF-κB signaling pathways in the retinas of Zc3h11a heterozygous knockout (Het-KO) mice.

(A-E) qRT-PCR quantification of ZC3H11A, PI3K, AKT, IκBα, and NF-κB mRNA in the retina (n=3 mice/group): Zc3h11a expression was decreased in Zc3h11a Het-KO mouse retinas, while PI3K and AKT expression were increased; IκBα expression was decreased and NF-κB expression was increased. (F-L) Western blot analysis of Zc3h11a, PI3K, p-AKT/AKT, IκBα, and NF-κB protein levels in the retina (n=3 mice/group). (F-I) Quantitative analysis of ZC3H11A and PI3K levels normalized to GAPDH, while p-AKT levels were normalized to AKT: ZC3H11A expression was decreased in Zc3h11a Het-KO mouse retinas, while PI3K and p-AKT/AKT expression were increased. (J-L) Quantitative analyses of IκBα and NF-κB normalized to GAPDH: IκBα expression was decreased in Zc3h11a Het-KO mouse retinas.

Elevated expression of TGF-β1, MMP-2, and IL-6 in retina and sclera of Zc3h11a Het-KO mice, with disrupted scleral ultrastructure.

(A, B) qRT-PCR quantification of TGF-β1, MMP-2 and IL-6 mRNA in retina (A) and sclera (B) (n=3 mice/group): Expression of TGF-β1, MMP-2 and IL-6 was increased in both retina and sclera of Zc3h11a Het-KO mice. (C) TEM structure of sclera, WT mice, organized collagen fibers with regular transverse/longitudinal arrangement. Zc3h11a Het-KO mice, disorganized collagen fibers structure with irregular arrangement (black arrows). (D) Quantitative analyses of TGF-β1 and MMP-2 normalized to GAPDH (n=3 mice/group): Expression of TGF-β1 and MMP-2 was increased in retinas of Zc3h11a Het-KO mice.

Mechanism of Zc3h11a haploinsufficiency-driven dysregulation of PI3K-AKT/NF-κB signaling in myopia pathogenesis.

NF-κB activation, loss of ZC3H11A impairs nuclear export of IκBα mRNA, leading to reduced cytoplasmic IκBα protein levels and consequent hyperactivation of NF-κB. PI3K-AKT activation. ZC3H11A deficiency upregulates PI3K, enhancing the conversion of PIP2 to PIP3, which drives AKT phosphorylation (p-AKT). Activated p-AKT promotes IκBα degradation, further amplifying NF-κB nuclear translocation and transcriptional activity. Downstream effects, the combined hyperactivity of PI3K-AKT and NF-κB pathways elevates the expression of: TGF-β1 (extracellular matrix remodeling), MMP-2 (collagen degradation), IL-6 (pro-inflammatory signaling), collectively driving scleral thinning and inflammatory responses in myopia pathogenesis.

Generation of Zc3h11a KO mice.

(A) Generation of Zc3h11a KO mice in C57BL/6J background using CRISPR/Cas9 technology: Exon 5 to exon 6 of the Zc3h11a transcript was used as the KO region; this region contains a 244 bp coding sequence, and knocking out this region will result in loss of protein function (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) There were no significant differences in fundus photographs between Zc3h11a Het-KO and WT mice, WT (A) and Het-KO mice (B) (n=3). (C. D) There were no significant differences in HE staining between Zc3h11a Het-KO and WT mice. WT (C) and Het-KO mice (D) (n=3).

Quantitative analysis of cone cells and rod cells in Zc3h11a Het-KO.

(A) No significance differences in Opsin-1 abundance. (B) No significance differences in Rhodopsin protein abundance.

The relative mRNA expression levels of IκBα in the nucleus.

Transfection of ZC3H11A overexpression mutant plasmid into cells resulted in a significant decrease in the mRNA expression levels of IκBα in four mutants (ZC3H11AV138I, ZC3H11AG43E, ZC3H11AP154L and ZC3H11AS747T).

Sequence of oligonucleotides