ZC3H11A mutations cause high myopia by triggering PI3K-AKT and NF-κB-mediated signaling pathway in humans and mice
- National Engineering Research Center of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, China
- National Clinical Research Center for Ocular Diseases, Eye Hospital, Wenzhou Medical University, China
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, China
Figures
Figure 1
Structural and evolutionary analysis of ZC3H11A mutations in high myopia.
(A) Genomic 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.
Figure 2 with 2 supplements
Zc3h11a+/- mice（n=14）exhibit myopic shifts in refractive parameters compared with WT mice（n=10）.
(A) Zc3h11a+/- mice showed myopia in diopter. (B, C) Axial length and vitreous chamber depth increased elongation in Zc3h11a+/- mice at weeks 4 and 6. (D–F) No genotype-dependent differences in anterior chamber depth, lens thickness, or body weight. Statistical significance of differences was assessed using independent-samples t-tests. The results are expressed as mean ± standard deviation (SD), with error bars representing the SD. p-Values are indicated as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
Figure 2—figure supplement 1
Generation of Zc3h11a+/- mice.
(A) Generation of Zc3h11a knockout (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+/- mice and wild-type mice.
Figure 2—figure supplement 2
Fundus photographs and HE staining of Zc3h11a+/- and wild-type (WT) mice at eighth week.
(A, B) There were no significant differences in fundus photographs between Zc3h11a+/- and WT mice, WT (A) and Zc3h11a+/- mice (B) (n=3). (C, D) There were no significant differences in HE staining between Zc3h11a+/- and WT mice. WT (C) and Zc3h11a+/- mice (D) (n=3).
Figure 3 with 1 supplement
Zc3h11a+/- mice exhibited bipolar cell dysfunction, accompanied by reduced abundance of the key bipolar cell marker protein PKC-α .
(A) Representative scotopic electroretinography (ERG) responses from Zc3h11a+/- and wild-type (WT) eyes at dark 0.01 (2.02 log cd·s/m2), dark 3.0 (0.48 log cd·s/m2), and 10.0 (0.98 log cd·s/m2). (B, C) Quantification of a-wave (photoreceptor function) and b-wave (bipolar cell function) amplitudes. Zc3h11a+/- 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, PKC-α (key bipolar cell marker protein), Opsin-1 (key cone photoreceptors marker protein), and Rhodopsin (key rod photoreceptors marker protein). (G, H) Zc3h11a and PKC-α protein abundance reduced in the retina of Zc3h11a+/- mice（n=3） compared with WT mice（n=3）. (I, J) Western blot analysis showed that the PKC-α protein content in the retina of Zc3h11a+/- mice (n=4) was decreased relative to WT retinas(n=4). Statistical significance of differences was assessed using independent-samples t-tests. The results are expressed as mean ± standard deviation (SD), with error bars representing the SD. p-Values are indicated as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
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Figure 3—source data 1
Original files used for the western blot analysis in Figure 3I.
- https://cdn.elifesciences.org/articles/91289/elife-91289-fig3-data1-v1.zip
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Figure 3—source data 2
Includes the original western blots for Figure 3I, with indicated relevant bands and groupings.
- https://cdn.elifesciences.org/articles/91289/elife-91289-fig3-data2-v1.zip
Figure 3—figure supplement 1
Quantitative immunofluorescence analysis of Opsin-1 and Rhodopsin protein levels in the retinas of Zc3h11a+/- and WT mice (n=3 mice/group).
(A) No significant differences in Opsin-1 abundance. (B) No significant differences in Rhodopsin protein abundance. Statistical significance of differences was assessed using independent-samples t-tests. The results are expressed as mean ± standard deviation (SD), with error bars representing the SD. p-Values are indicated as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
Figure 4
Transmission electron microscopy (TEM) showing the retinal abnormalities ultrastructure of Zc3h11a+/- mice.
(A–B) Inner nuclear layer (INL). (A) Wild-type (WT) mice, normal bipolar cell morphology; (B) Zc3h11a+/- 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+/- mice, no significant differences in photoreceptor nucleus morphology. (E–H) Photoreceptor membrane discs (MB). (E–F) WT mice, tightly stacked, uniformly arranged MB. (G–H) Zc3h11a+/- structural disruptions include the outer layer falls off, the local distribution is sparse, and the arrangement is chaotic and loose (red arrow).
Figure 5
RNA sequence reveals pathway dysregulation in the retina of Zc3h11a+/- mice.
(A) Volcano plot of differentially expressed genes (DEGs): 769 total (303 upregulated, 466 downregulated in Zc3h11a+/- (n=3) vs wild-type [WT] (n=3) 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.
Figure 6 with 1 supplement
Dysregulation of PI3K-AKT and NF-κB signaling pathways in the retinas of Zc3h11a+/- 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+/- mouse retinas, while PI3K and AKT expression levels 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+/- mouse retinas, while PI3K and p-AKT/AKT expression levels were increased. (J–L) Quantitative analyses of IκBα and NF-κB normalized to GAPDH: IκBα expression was decreased in Zc3h11a+/- mouse retinas. Statistical significance of differences was assessed using independent-samples t-tests. The results are expressed as mean ± standard deviation (SD), with error bars representing the SD. p-Values are indicated as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
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Figure 6—source data 1
Original data files for the Zc3h11a western blot analysis presented in Figure 6F.
- https://cdn.elifesciences.org/articles/91289/elife-91289-fig6-data1-v1.zip
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Figure 6—source data 2
Includes the original western blot membrane for Zc3h11a protein in Figure 6F, with clearly marked target bands and experimental groupings.
- https://cdn.elifesciences.org/articles/91289/elife-91289-fig6-data2-v1.zip
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Figure 6—source data 3
Original data files for the PI3K western blot analysis presented in Figure 6F.
- https://cdn.elifesciences.org/articles/91289/elife-91289-fig6-data3-v1.zip
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Figure 6—source data 4
Includes the original western blot membrane for PI3K protein in Figure 6F, with clearly marked target bands and experimental groupings.
- https://cdn.elifesciences.org/articles/91289/elife-91289-fig6-data4-v1.zip
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Figure 6—source data 5
Original data files for the AKT western blot analysis presented in Figure 6F.
- https://cdn.elifesciences.org/articles/91289/elife-91289-fig6-data5-v1.zip
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Figure 6—source data 6
Includes the original western blot membrane for AKT protein in Figure 6F, with clearly marked target bands and experimental groupings.
- https://cdn.elifesciences.org/articles/91289/elife-91289-fig6-data6-v1.zip
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Figure 6—source data 7
Original data files for the p-AKT western blot analysis presented in Figure 6F.
- https://cdn.elifesciences.org/articles/91289/elife-91289-fig6-data7-v1.zip
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Figure 6—source data 8
Includes the original western blot membrane for p-AKT protein in Figure 6F, with clearly marked target bands and experimental groupings.
- https://cdn.elifesciences.org/articles/91289/elife-91289-fig6-data8-v1.zip
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Figure 6—source data 9
Original data files for the IκBα western blot analysis presented in Figure 6J.
- https://cdn.elifesciences.org/articles/91289/elife-91289-fig6-data9-v1.zip
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Figure 6—source data 10
Includes the original western blot membrane for IκBα protein in Figure 6J, with clearly marked target bands and experimental groupings.
- https://cdn.elifesciences.org/articles/91289/elife-91289-fig6-data10-v1.zip
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Figure 6—source data 11
Original data files for the NF-κB western blot analysis presented in Figure 6J.
- https://cdn.elifesciences.org/articles/91289/elife-91289-fig6-data11-v1.zip
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Figure 6—source data 12
Includes the original western blot membrane for NF-κB protein in Figure 6J, with clearly marked target bands and experimental groupings.
- https://cdn.elifesciences.org/articles/91289/elife-91289-fig6-data12-v1.zip
Figure 6—figure supplement 1
The relative mRNA expression levels of IκBα in the nucleus (n=3/group).
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). Statistical significance of differences was assessed using independent-samples t-tests. The results are expressed as mean ± standard deviation (SD), with error bars representing the SD. p-Values are indicated as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
Figure 7
Elevated expression of TGF-β1, MMP-2, and IL-6 in retina and sclera of Zc3h11a+/- 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+/- mice. (C) Transmission electron microscopy (TEM) structure of sclera, wild-type (WT) mice, organized collagen fibers with regular transverse/longitudinal arrangement. Zc3h11a+/- 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+/- mice. Statistical significance of differences was assessed using independent-samples t-tests. The results are expressed as mean ± standard deviation (SD), with error bars representing the SD. p-Values are indicated as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
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Figure 7—source data 1
Original data files for the MMP-2 western blot analysis presented in Figure 7D.
- https://cdn.elifesciences.org/articles/91289/elife-91289-fig7-data1-v1.zip
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Figure 7—source data 2
Includes the original western blot membrane for MMP-2 protein in Figure 7D, with clearly marked target bands and experimental groupings.
- https://cdn.elifesciences.org/articles/91289/elife-91289-fig7-data2-v1.zip
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Figure 7—source data 3
Original data files for the TGF-β1 western blot analysis presented in Figure 7D.
- https://cdn.elifesciences.org/articles/91289/elife-91289-fig7-data3-v1.zip
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Figure 7—source data 4
Includes the original western blot membrane for TGF-β1 protein in Figure 7D, with clearly marked target bands and experimental groupings.
- https://cdn.elifesciences.org/articles/91289/elife-91289-fig7-data4-v1.zip
Figure 8
Mechanism of Zc3h11a haploinsufficiency-driven dysregulation of PI3K-AKT/NF-κB signaling in myopia pathogenesis.
Loss of ZC3H11A impairs nuclear export of IκBα mRNA, leading to reduced cytoplasmic IκBα protein levels and consequent hyperactivation of NF-κB, and also 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, 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.
Author response image 1
Tables
Table 1
The clinical features and mutations of affected patients.
| Patient information | Refraction (D) | Axial length (mm) | Variation information | Prediction software and databases | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ID | Sex | Age | OD | OS | OD | OS | Genotype | chromosomal positions | Existing_variation (rs numbers) | SIFT (score) | PolyPhen2 (score) | CADD (score) | gnomAD | Clinvar |
| 1 | M | 18 | –6.50 | –5.125 | 25.68 | 25.34 | c.412G>A:p. Val138Ile, Het | chr1:203798692–203798692 | rs142418357 | D (0.03) | PB (0.912) | 24.3 | 7.95E–06 | – |
| 2 | F | 15 | –4.25 | –6.125 | 24.61 | 25.11 | c.128G>A:p. Gly43Glu, Het | chr1:203787771–203787771 | – | D (0) | PS (0.906) | 26 | – | – |
| 3 | M | 17 | –8.00 | –8.75 | 26.16 | 27.02 | c.461C>T:p. Pro154Leu, Het | chr1:203798741–203798741 | – | D (0) | PS (0.628) | 29.3 | – | – |
| 4 | F | 15 | –6.25 | –3.70 | 25.53 | 24.86 | c.2239T>A:p. Ser747Thr, Het | chr1:203821333–203821333 | – | T (0.1) | PS (0.838) | 22.7 | – | – |
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M, male; F, female; Het, heterozygote; OD, ocular dexter; OS, oculus sinister; D, deleterious, T, tolerated; PB, probably_damaging; PS, possibly_damaging; gnomAD, Genome Aggregation Database; AF, allele frequency; –, not applicable.
Additional files
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Supplementary file 1
Sequence of oligonucleotides.
- https://cdn.elifesciences.org/articles/91289/elife-91289-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/91289/elife-91289-mdarchecklist1-v1.pdf
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ZC3H11A mutations cause high myopia by triggering PI3K-AKT and NF-κB-mediated signaling pathway in humans and mice
eLife 12:RP91289.
https://doi.org/10.7554/eLife.91289.4
