ZC3H11A mutations cause high myopia by triggering PI3K-AKT and NF-κB mediated signaling pathway in humans and mice

Reviewer #2 (Public review):

Summary:

The authors reported that mutations were identified in the ZC3H11A gene in four adolescents from 1015 high myopia subjects in their myopia cohort. They further generated Zc3h11a knockout mice utilizing the CRISPR/Cas9 technology.

Comments on revisions:

Chong Chen and colleagues revised the manuscript; however, none of my suggestions from the initial review have been sufficiently addressed.

(1) I indicated that the pathogenicity and novelty of the mutation need to be determined according to established guidelines and databases. However, the conclusion was still drawn without sufficient justification.
(2) The phenotype of heterozygous mutant mice is too weak to support the gene's contribution to high myopia. The revised manuscript does not adequately address these discrepancies. Furthermore, no explanation was provided for why conditional gene deletion was not used to avoid embryonic lethality, nor was there any discussion on tissue- or cell-specific mechanistic investigations.
(3) The title, abstract, and main text continue to misrepresent the role of the inflammatory intracellular PI3K-AKT and NF-κB signaling cascade in inducing high myopia. No specific cell types have been identified as contributors to the phenotype. The mice did not develop high myopia, and no relationship between intracellular signaling and myopia progression has been demonstrated in this study.

Reviewer #3 (Public review):

Chen et al have identified a new candidate gene for high myopia, ZC3H11A, and using a knock-out mouse model, have attempted to validate it as a myopia gene and explain a potential mechanism. They identified 4 heterozygous missense variants in highly myopic teenagers. These variants are in conserved regions of the protein, and predicted to be damaging, but the only evidence the authors provide that these specific variants affect protein function is a supplement figure showing decreased levels of IκBα after transfection with overexpression plasmids (not specified what type of cells were transfected). This does not prove that these mutations cause loss of function, in fact it implies they have a gain-of-function mechanism. They then created a knock-out mouse. Heterozygotes show myopia at all ages examined but increased axial length only at very early ages. Unfortunately, the authors do not address this point or examine corneal structure in these animals. They show that the mice have decreased B-wave amplitude on electroretinogram (a sign of retinal dysfunction associated with bipolar cells), and decreased expression of a bipolar cell marker, PKCα. On electron microscopy, there are morphologic differences in the outer nuclear layer (where bipolar, amacrine, and horizontal cell bodies reside). Transcriptome analysis identified over 700 differentially expressed genes. The authors chose to focus on the PI3K-AKT and NF-κB signaling pathways and show changes in expression of genes and proteins in those pathways, including PI3K, AKT, IκBα, NF-κB, TGF-β1, MMP-2 and IL-6, although there is very high variability between animals. They propose that myopia may develop in these animals either as a result of visual abnormality (decreased bipolar cell function in the retina) or by alteration of NF-κB signaling. These data provide an interesting new candidate variant for development of high myopia, and provide additional data that MMP2 and IL6 have a role in myopia development. For this revision, none of my previous suggestions have been addressed.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

Chen and colleagues investigated ZC3H11A as a potential cause of high myopia (HM) in humans through the analysis of exome sequencing in 1,015 adolescents and experiments involving Zc3h11a knock-out mice. The authors showed four possibly pathogenic missense variants in four adolescents with HM. After that, the authors presented the phenotypic features of Zc3h11a knock-out mice, the result of RNA-sequencing, and a comparison of mRNA and protein levels of the functional candidates between wild-type and Zc3h11a knock-out mice. Based on their observations, the authors concluded that ZC3H11A protein contributes to the early onset of myopia.

The strengths of this manuscript include: (1) successful identification of characteristic ophthalmic phenotypes in Zc3h11a knock-out mice, (2) demonstration of biological features related to myopia, such as PI3K-AKT and NF-kB pathways, and (3) inclusion of supporting human genetic data in individuals with HM. On the other hand, the weaknesses of this paper appear to be: (1) the lack of robust evidence from their genomic analysis, and (2) insufficient evidence to support phenotypic similarity between humans with ZC3H11A mutations and Zc3h11a knock-out mice. Given that the biological mechanisms of high myopia are not fully understood, the identification of a novel gene is valuable. As described in the manuscript, it is worth noting that the previous study using myopic mouse model has implicated the role of ZC3H11A in the etiology of myopia (Fan et al. Plos Genet 2012).

Thank you very much for your valuable suggestions.

Specific comments:

(1) I am concerned about the certainty of similarity in phenotypes between individuals with ZC3H11A mutation and Zc3h11a knock-out mice. A crucial point would be that there are no statistical differences in axial lengths (ALs) between wild-type and Zc3h11a knock-out mice at 8W and 10W, even though ALs in the individuals with ZC3H11A mutation were long. I would also like to note that the phenotypic information of these individuals is not available in the manuscript, although the authors indicated the suppressed b-wave amplitude in Zc3h11a knock-out mice. Considering that the authors described that "Detailed ophthalmic examinations were performed (lines: 321-323)", the detailed clinical features of these individuals should be included in the manuscript.

Thank you for your valuable comments. The axial length in Zc3h11a Het-KO mice were found to be significantly greater than in WT littermates at weeks 4 and 6 (Independent samples t-test, p<0.05; Figure 2A and B). Although no significant differences were observed at other time points, there was still some degree of increase in these parameters. We continued to measure corneal curvature and found no significant differences between the two groups. Therefore, the difference in refraction may be due to the small size of the mouse eye. A 1 D change in refraction corresponds to only a 5-6 μm change in AL(1). However, the SD-OCT resolution used in this study is relatively low (theoretical resolution of 6 μm)(2, 3), so the small changes measured in vitreous cavity depth and AL may not be statistically significant. Additionally, some studies have shown that axial lengths reported in frozen sections are longer than those measured in vivo for age-matched mice(1, 4). Another possible explanation is that the curvature and refractive power of the lens have changed. These hypotheses provide a reasonable explanation for the mismatch between changes in refraction and ocular length parameters.

Reference

(1) Schmucker C, Schaeffel F. A paraxial schematic eye model for the growing C57BL/6 mouse. Vision research 44, 1857-1867 (2004).

(2) Yuan Y, Chen F, Shen M, Lu F, Wang J. Repeated measurements of the anterior segment during accommodation using long scan depth optical coherence tomography. Eye & contact lens 38, 102-108 (2012).

(3) Shen M, et al. SD-OCT with prolonged scan depth for imaging the anterior segment of the eye. Ophthalmic Surgery, Lasers and Imaging Retina 41, S65-S69 (2010).

(4) Schmucker C, Schaeffel F. In vivo biometry in the mouse eye with low coherence interferometry. Vision research 44, 2445-2456 (2004).

Additionally, regarding the “detailed ophthalmic examinations”, due to our patients were selected from a myopia screening cohort of over one million (children and adolescents myopia survey [CAMS] program), and ophthalmic examination only includes semi-annual refractive error measurements (a total of 5 times, with refractive error being the average of the three maximum values) and only one axial length measurement. The inappropriate description of “Detailed clinical features” has been removed.

(2) The term "pathogenic variant" should be used cautiously. Please clarify the pathogenicity of the reported variants in accordance with the ACMG guideline.

Thank you for your valuable comments. Four missense mutations in the ZC3H11A gene (c.412G>A, p.V138I; c.128G>A, p.G43E; c.461C>T, p.P154L; and c.2239T>A, p.S747T) were identified in the 1015 HM patients aged from 15 to 18 years. All of the identified mutations exhibited very low frequencies or does not exist in the Genome Aggregation Database (gnomAD) and Clinvar, and using pathogenicity prediction software SIFT, PolyPhen2, and CADD, most of them display high pathogenicity levels. Among them, c.412G>A, c.128G>A and c.461C>T were located in or around a domain named zf-CCCH_3 (Figure 1A and B). Furthermore, all of the mutation sites were located in highly conserved amino acids across different species (Figure 1C). Four mutations resulted in a higher degree of conformational flexibility and altered the negative charge at the corresponding sites (Figure 1D and E). Meanwhile, through transfection of overexpression mutant plasmids, it was found that compared to the wild-type, the mRNA expression levels of IκBα in the nucleus of all four mutant types (ZC3H11A^V138I, ZC3H11A^G43E, ZC3H11A^P154L and ZC3H11A^S747T) were significantly reduced (Supplement Figure 3). According to the ACMG guidelines, the above mutations can be classified as “Pathogenic Moderate”.

(3) The genetic analysis does not fully support the claim that ZC3H11A is causative for HM. While the authors showed the rare allele frequencies and high CADD scores (> 20) of the identified variants, these were insufficient to establish causality. A helpful way to assess the causality would be performing a segregation analysis. An alternative approach is to show significant association by performing a gene-level association test. Assessing the pathogenicity of the variants using various prediction software, such as SIFT, PolyPhen2, and REVEL may also provide additional supportive evidence.

Thank you for your valuable comments. We have addad the pathogenicity of the variants using various prediction software, such as SIFT, PolyPhen2, CADD, and the population variation databases, such as Genome Aggregation Database (gnomAD_AF) and ClinVar. Meanwhile, through transfection of overexpression mutant plasmids, it was found that compared to the wild-type, the mRNA expression levels of IκBα in the nucleus of all four mutant types (ZC3H11A^V138I, ZC3H11A^G43E, ZC3H11A^P154L and ZC3H11A^S747T) were significantly reduced (Supplement Figure 3).

(4) As shown in Figure 2, significant differences in refraction were observed from 4 weeks to 10 weeks. Nevertheless, no differences were observed in AL, anterior/vitreous chamber depth, and lens depth. The author should experimentally clarify what factors contribute to the observed difference in refraction.

Thank you for your valuable comments. The existing data show significant differences in refraction between 4 and 10 weeks, with the AL and vitreous cavity depth of Het mice being longer than those of WT mice at 4 and 6 weeks. Although no significant differences were observed at other time points, there was still some degree of increase in these parameters. We continued to measure corneal curvature and found no significant differences between the two groups. Therefore, the difference in refraction may be due to the small size of the mouse eye. A 1 D change in refraction corresponds to only a 5-6 μm change in AL(1). However, the SD-OCT resolution used in this study is relatively low (theoretical resolution of 6 μm)(2, 3), so the small changes measured in vitreous cavity depth and AL may not be statistically significant. Additionally, some studies have shown that axial lengths reported in frozen sections are longer than those measured in vivo for age-matched mice(1, 4). Another possible explanation is that the curvature and refractive power of the lens have changed. These hypotheses provide a reasonable explanation for the mismatch between changes in refraction and ocular length parameters.

Reference

(1) Schmucker C, Schaeffel F. A paraxial schematic eye model for the growing C57BL/6 mouse. Vision research 44, 1857-1867 (2004).

(2) Yuan Y, Chen F, Shen M, Lu F, Wang J. Repeated measurements of the anterior segment during accommodation using long scan depth optical coherence tomography. Eye & contact lens 38, 102-108 (2012).

(3) Shen M, et al. SD-OCT with prolonged scan depth for imaging the anterior segment of the eye. Ophthalmic Surgery, Lasers and Imaging Retina 41, S65-S69 (2010).

(4) Schmucker C, Schaeffel F. In vivo biometry in the mouse eye with low coherence interferometry. Vision research 44, 2445-2456 (2004).

(5) The gene names should be italicized throughout the manuscript.

Thank you for your valuable comments. The gene names have been italicized throughout the manuscript.

(6) Table 1: providing chromosomal positions and rs numbers (if available) would be helpful for readers.

Thank you for your valuable comments. We have provided the chromosome positions and rs number (if available) of each mutation in Table 1.

(7) Figure 5b, c, and d: the results of pathway analysis and GO enrichment analysis are difficult to interpret due to the small font size. It would be preferable to present these results in tables. Moreover, the authors should set a significant threshold in the enrichment analyses.

Thank you for your valuable comments. We have adjusted the font size of the image. In the retina transcriptome analysis, we have set Fold change (FC) of at least two and a P value < 0.05 as thresholds to analyze differentially expressed genes (DEGs). The GO terms and KEGG pathways enrichment analysis selected the top 20 with the most significant differences or the highest number of enriched genes for display.

Reviewer #2 (Public Review):

Summary: Chong Chen and colleagues reported that mutations were identified in the ZC3H11A gene in four adolescents from 1015 high myopia subjects in their myopia cohort. They further generated Zc3h11a knockout mice utilizing the CRISPR/Cas9 technology. They analyzed the heterozygotes knockout mice compared to control littermates and found refractive error changes, electrophysiological differences, and retinal inflammation-related gene expression differences. They concluded that ZC3H11A may play a role in the early onset of myopia by regulating inflammatory responses.

Strengths:

Data were shown from both clinical cohort and animal models.

Weaknesses:

Their findings are interesting and important, however; they need to resolve several points to make the current conclusion.

(1) They described the ZC3H11A gene as a pathogenic variant for high myopia. It should be classified as pathogenic according to the guidelines of the American College of Medical Genetics and Genomics (Richards et al., Genet Med 17(5):405-24, 2015). The modes of inheritance for the families need to be shown. They also described identifying the gene as a "new" candidate. It should be checked in databases such as gnomAD and ClinVar, and any previous publications and be declared as a novel variant.

Thank you for your valuable comments. Four missense mutations in the ZC3H11A gene (c.412G>A, p.V138I; c.128G>A, p.G43E; c.461C>T, p.P154L; and c.2239T>A, p.S747T) were identified in the 1015 HM patients aged from 15 to 18 years. All of the identified mutations exhibited very low frequencies or does not exist in the Genome Aggregation Database (gnomAD) and Clinvar, and using pathogenicity prediction software SIFT, PolyPhen2, and CADD, most of them display high pathogenicity levels. Among them, c.412G>A, c.128G>A and c.461C>T were located in or around a domain named zf-CCCH_3 (Figure 1A and B). Furthermore, all of the mutation sites were located in highly conserved amino acids across different species (Figure 1C). Four mutations resulted in a higher degree of conformational flexibility and altered the negative charge at the corresponding sites (Figure 1D and E). Meanwhile, through transfection of overexpression mutant plasmids, it was found that compared to the wild-type, the mRNA expression levels of IκBα in the nucleus of all four mutant types (ZC3H11A^V138I, ZC3H11A^G43E, ZC3H11A^P154L and ZC3H11A^S747T) were significantly reduced (Supplement Figure 3). According to the ACMG guidelines, the above mutations can be classified as “Pathogenic Moderate”.

Unfortunately, our patients are part of the MAGIC project (aged 15 years or older), a cohort consists of thousands of individuals with HM (patients from the children and adolescents myopia survey [CAMS] program) who have undergone WES, and their parents' relevant information was not collected for performing a segregation analysis.

(2) The phenotypes of the heterozygote mice are weak overall. The het mice showed mild to moderate myopic refractive shifts from 4 to 10 weeks of age. However, this cannot be explained by other ocular biometrics such as anterior chamber depth or lens thickness. Some differences are found between het and WT littermates in axial length and vitreous chamber depth but disappear after 8 weeks old. Furthermore, the early differences are not enough to explain the refractive error changes. They mentioned that they did not use homozygotes because of the embryonic lethality. I would strongly suggest employing conditional knockout systems to analyze homozygotes. This will also be able to identify the causative tissues/cells because they assume bipolar cells are functional. The cells in the retinal pigment epithelium and choroid are also important to contribute to myopia development.

Thank you for your valuable comments. The existing data show significant differences in refraction between 4 and 10 weeks, with the AL and vitreous cavity depth of Het mice being longer than those of WT mice at 4 and 6 weeks. Although no significant differences were observed at other time points, there was still some degree of increase in these parameters. We continued to measure corneal curvature and found no significant differences between the two groups. Therefore, the difference in refraction may be due to the small size of the mouse eye. A 1 D change in refraction corresponds to only a 5-6 μm change in AL(1). However, the SD-OCT resolution used in this study is relatively low (theoretical resolution of 6 μm)(2, 3), so the small changes measured in vitreous cavity depth and AL may not be statistically significant. Additionally, some studies have shown that axial lengths reported in frozen sections are longer than those measured in vivo for age-matched mice(1, 4). Another possible explanation is that the curvature and refractive power of the lens have changed. These hypotheses provide a reasonable explanation for the mismatch between changes in refraction and ocular length parameters.

Reference

(1) Schmucker C, Schaeffel F. A paraxial schematic eye model for the growing C57BL/6 mouse. Vision research 44, 1857-1867 (2004).

(2) Yuan Y, Chen F, Shen M, Lu F, Wang J. Repeated measurements of the anterior segment during accommodation using long scan depth optical coherence tomography. Eye & contact lens 38, 102-108 (2012).

(3) Shen M, et al. SD-OCT with prolonged scan depth for imaging the anterior segment of the eye. Ophthalmic Surgery, Lasers and Imaging Retina 41, S65-S69 (2010).

(4) Schmucker C, Schaeffel F. In vivo biometry in the mouse eye with low coherence interferometry. Vision research 44, 2445-2456 (2004).

The drawback is that, we did not conduct relevant research on homozygous knockout mice. The first reason is that our patient's mutation pattern is heterozygous mutation (Heterozygous knockout mice can better simulate human phenotypes). The second reason is that homozygous knockout mice are lethal, and we did not use the conditional knockout mouse model for further research. At the same time, we limited the pathway of myopia to the recognized and classical retina-sclera pathway, and did not study other pathways such as retinal pigment epithelium and choroid.

(3) Their hypothesis regarding inflammatory gene changes and myopic development is not logical. Are the inflammatory responses evoked from bipolar cells? Did the mice show an accumulation of inflammatory cells in the inner retina? Visible retinal inflammation is not generally seen in either early-onset or high-myopia human subjects. Can this be seen in the actual subjects in the cohort? To me, this is difficult to adapt the retina-to-sclera signaling they mentioned in the discussion so far. Egr-1 may be examined as described.

Thank you for your valuable comments. We have removed the hypothesis regarding inflammatory gene changes and myopic development. At present, the explanation is based solely on the correlation of signal pathways, the theoretical basis comes from the reference literature:

“Lin et al., Role of Chronic Inflammation in Myopia Progression: Clinical Evidence and Experimental Validation. EBioMedicine, 2016 Aug:10:269-81, Figure 7.”

Reviewer #3 (Public Review):

Chen et al have identified a new candidate gene for high myopia, ZC3H11A, and using a knock-out mouse model, have attempted to validate it as a myopia gene and explain a potential mechanism. They identified 4 heterozygous missense variants in highly myopic teenagers. These variants are in conserved regions of the protein, but the authors provide no evidence that these specific variants affect protein function. They then created a knock-out mouse. Heterozygotes show myopia at all ages examined but increased axial length only at very early ages. Unfortunately, the authors do not address this point or examine corneal structure in these animals. They show that the mice have decreased B-wave amplitude on electroretinogram (a sign of retinal dysfunction associated with bipolar cells), and decreased expression of a bipolar cell marker, PKCa. They do not address, however, whether there are fewer bipolar cells, or simply decreased expression of the marker protein. On electron microscopy, there are morphologic differences in the outer nuclear layer (where bipolar, amacrine, and horizontal cell bodies reside). Transcriptome analysis identified over 700 differentially expressed genes. The authors chose to focus on the PI3K-AKT and NF-kB signaling pathways and show changes in the expression of genes and proteins in those pathways, including PI3K, AKT, IkBa, NF-kB, TGF-b1, MMP-2, and IL-6, although there is very high variability between animals. They propose that myopia may develop in these animals either as a result of visual abnormality (decreased bipolar cell function in the retina) or by alteration of NF-kB signaling. These data provide an interesting new candidate variant for the development of high myopia, and provide additional data that MMP2 and IL6 have a role in myopia development, but do not support the claim of the title that myopia is caused by an inflammatory reaction.

Thank you for your valuable comments. Four missense mutations in the ZC3H11A gene (c.412G>A, p.V138I; c.128G>A, p.G43E; c.461C>T, p.P154L; and c.2239T>A, p.S747T) were identified in the 1015 HM patients aged from 15 to 18 years. All of the identified mutations exhibited very low frequencies or does not exist in the Genome Aggregation Database (gnomAD) and Clinvar, and using pathogenicity prediction software SIFT, PolyPhen2, and CADD, most of them display high pathogenicity levels. Among them, c.412G>A, c.128G>A and c.461C>T were located in or around a domain named zf-CCCH_3 (Figure 1A and B). Furthermore, all of the mutation sites were located in highly conserved amino acids across different species (Figure 1C). Four mutations resulted in a higher degree of conformational flexibility and altered the negative charge at the corresponding sites (Figure 1D and E). Meanwhile, through transfection of overexpression mutant plasmids, it was found that compared to the wild-type, the mRNA expression levels of IκBα in the nucleus of all four mutant types (ZC3H11A^V138I, ZC3H11A^G43E, ZC3H11A^P154L and ZC3H11A^S747T) were significantly reduced (Supplement Figure 3). According to the ACMG guidelines, the above mutations can be classified as “Pathogenic Moderate”.

The existing data show significant differences in refraction between 4 and 10 weeks, with the AL and vitreous cavity depth of Het mice being longer than those of WT mice at 4 and 6 weeks. Although no significant differences were observed at other time points, there was still some degree of increase in these parameters. We continued to measure corneal curvature and found no significant differences between the two groups. Therefore, the difference in refraction may be due to the small size of the mouse eye. A 1 D change in refraction corresponds to only a 5-6 μm change in AL(1). However, the SD-OCT resolution used in this study is relatively low (theoretical resolution of 6 μm)(2, 3), so the small changes measured in vitreous cavity depth and AL may not be statistically significant. Additionally, some studies have shown that axial lengths reported in frozen sections are longer than those measured in vivo for age-matched mice(1, 4). Another possible explanation is that the curvature and refractive power of the lens have changed. These hypotheses provide a reasonable explanation for the mismatch between changes in refraction and ocular length parameters.

To evaluate the change in the number of a specific type of retinal cells, the most commonly used experimental method involves staining with antibodies specific to the target cell type, followed by fluorescence microscopy. The fluorescence intensity or the number of cells can be analyzed semi-quantitatively to assess the changes in the specific cell type in the retina. For example, in retinal degenerative models, rhodopsin-specific staining is used to identify the loss of rod cells. In our study, we selected PCK-α as a marker protein for bipolar cells to assess their number. Additionally, transmission electron microscopy (TEM) was used to observe damage to the cell morphology in the inner nuclear layer (INL) of Het mice, where bipolar cell bodies are located. Based on both sets of data, we conclude that bipolar cells have indeed undergone structural damage and a reduction in number.

Reference

(1) Schmucker C, Schaeffel F. A paraxial schematic eye model for the growing C57BL/6 mouse. Vision research 44, 1857-1867 (2004).

(2) Yuan Y, Chen F, Shen M, Lu F, Wang J. Repeated measurements of the anterior segment during accommodation using long scan depth optical coherence tomography. Eye & contact lens 38, 102-108 (2012).

(3) Shen M, et al. SD-OCT with prolonged scan depth for imaging the anterior segment of the eye. Ophthalmic Surgery, Lasers and Imaging Retina 41, S65-S69 (2010).

(4) Schmucker C, Schaeffel F. In vivo biometry in the mouse eye with low coherence interferometry. Vision research 44, 2445-2456 (2004).

We have removed the hypothesis regarding inflammatory gene changes and myopic development. At present, the explanation is based solely on the correlation of signal pathways, the theoretical basis comes from the reference literature:

“Lin et al., Role of Chronic Inflammation in Myopia Progression: Clinical Evidence and Experimental Validation. EBioMedicine, 2016 Aug:10:269-81, Figure 7.”