A mouse model of brittle cornea syndrome caused by mutation in Zfp469

ABSTRACT Brittle cornea syndrome (BCS) is a rare recessive condition characterised by extreme thinning of the cornea and sclera. BCS results from loss-of-function mutations in the poorly understood genes ZNF469 or PRDM5. In order to determine the function of ZNF469 and to elucidate pathogenic mechanisms, we used genome editing to recapitulate a human ZNF469 BCS mutation in the orthologous mouse gene Zfp469. Ophthalmic phenotyping showed that homozygous Zfp469 mutation causes significant central and peripheral corneal thinning arising from reduced stromal thickness. Expression of key components of the corneal stroma in primary keratocytes from Zfp469BCS/BCS mice is affected, including decreased Col1a1 and Col1a2 expression. This alters the collagen type I/collagen type V ratio and results in collagen fibrils with smaller diameter and increased fibril density in homozygous mutant corneas, correlating with decreased biomechanical strength in the cornea. Cell-derived matrices generated by primary keratocytes show reduced deposition of collagen type I, offering an in vitro model for stromal dysfunction. Work remains to determine whether modulating ZNF469 activity will have therapeutic benefit in BCS or in conditions such as keratoconus in which the cornea thins progressively. This article has an associated First Person interview with the first author of the paper.


Introduction
Brittle Cornea Syndrome (BCS; MIM 229200, MIM 614170) is a rare autosomal recessive disorder that is characterised by extreme thinning of the cornea and sclera. Visual impairment may initially be a result of myopia and progressive keratoconus or keratoglobus but, as the name suggests, the thin and fragile corneas of affected individuals are prone to rupture leading to irreversible blindness (Al-Hussain et al., 2004). Also classified as a subtype of Ehlers-Danlos syndrome (EDS type VIB), this devastating condition often leads to general connective tissue dysfunction with skin hyperelasticity, joint hyperflexibility and, in approximately one third of cases, hearing impairment (Malfait et al., 2017).
BCS results from biallelic loss-of-function (LOF) mutations in ZNF469 or PRDM5 (Abu et al., 2008;Burkitt Wright et al., 2011). Mutations in these genes appear to cause an indistinguishable disorder, suggesting that they contribute to the same biological pathways. PRDM5 (PR/SET Domain 5), a widely expressed transcription factor that modulates development and maintenance (Duan et al., 2007;Meani et al., 2009;Porter et al., 2015) is known to play an important role in extracellular matrix (ECM) production by several tissues including skin fibroblasts (Porter et al., 2015) and in bone (Galli et al., 2012). The role played by ZNF469 in the healthy cornea and in BCS is less clear as the function of the very large protein encoded by this gene is poorly characterised. Since mutations in ZNF469 were first reported to cause BCS (Abu et al., 2008), 30 pathogenic compound heterozygous or homozygous mutations have been identified in the single coding exon of ZNF469 spanning 13 kilobases on chromosome 16q24.2 (Abu et al., 2008;Al-Owain et al., 2012;Christensen et al., 2010;1A), and, with the exception of one large deletion encompassing the gene (Ramappa et al., 2014), share common consequences of truncating the protein or, in the case of two missense mutations, to modify key residues involved in the coordination of zinc in the sixth C2H2 ZF domain. Fewer LOF mutations than expected have been seen in human populations (probability of being loss-of-function intolerant, pLI, = 0.72 (observed/expected = 0.2 (0.12 -0.37, 90% Confidence Interval) in gnomAD (Lek et al., 2016). Only four of the BCS mutations reported in affected individuals, p.Gln2149Serfs*51, p.Gln2149Alafs*42, p.Arg3442Glyfs*59 and p.Pro3584Glnfs*136, have been identified in a small number of heterozygous individuals in gnomAD v3.1 ( Table 1).
The orthologous gene in mouse, Zfp469 or Gm22, is located on chromosome 8 (NM_001362883). Clustal Omega (Sievers et al., 2011) was used to align orthologous protein sequences from mouse (Zfp469, NP_001354553.1 ) and human (ZNF469, NP_001354553.1), with 47% amino acid identity across the full-length of each protein (human 3953 amino acids, mouse 3765 amino acids). The 8 predicted C2H2 ZF domains (positions obtained from the SMART database (Letunic et al., 2020)) show a much higher degree of conservation (Fig.1B), consistent with ZF domains having an important functional role. Of the human BCS mutations, only some affected conserved amino acids ( Table 1). One of these (human p.Gly677*, mouse p.Gly634) was selected as the target for genome editing using CRISPR-Cas9n to create a mouse model of BCS.

Gene editing Zfp469 to generate Zfp469 BCS mice
In order to elucidate the role of Zfp469 in mice, genome editing was performed to recapitulate a human BCS mutation by creating a premature stop codon early in Zfp469 the C2H2 ZF domains.
Three pairs of sgRNA (Table S1) designed to target sequence encoding Zpf469 p.Gly634 were cloned into pX458 (pSpCas9(BB)-2A-GFP) (Ran et al., 2013) and tested for cleavage efficiency in mouse embryonic fibroblasts (MEFs). The pair of sgRNAs with the highest cleavage efficiency (data not shown) was subsequently in vitro transcribed, and purified RNA was injected, along with SpCas9n mRNA and the ssODN repair template, into C57Bl/6J mouse zygotes. Successful genome editing and repair inserted an in-frame V5 tag and generated a premature stop codon to truncate Zfp469 ( Fig.2A). Homozygous mutant mice were significantly smaller than their sex-matched litter-mate controls at 3 months of age ( Fig.2B and 2C), weighing 15-20% less than wildtype mice (males, p=0.003, females p=0.0429, one way ANOVA with Dunnett's multiple comparison test). Body length was decreased by approximately 5% in Zfp469 BCS/BCS mice relative to wildtype age-and sex-matched littermates, but this difference was not statistically significant at 3 or 6 months of age (data not shown). There was no difference in eye size, measured using manual calipers, between genotypes in females at 3 months of age (average eye length from the front of the cornea to the posterior: +/+ 3.34 ± 0.13 mm (s.d., n=3), BCS/BCS 3.33 ± 0.13 mm (s.d., n=2). Other than the reduction in body weight, heterozygous and homozygous Zfp469 BCS mice were viable and fertile, with each genotype arising in the expected frequencies from heterozygote x heterozygote crosses (Table S2).
Expression of Zfp469 in corneal keratocytes freshly isolated from corneas pooled by genotype was assessed by RT-qPCR using primers specific for Zfp469 or for the V5-tag insertion.
Relative Zfp469 expression was increased to 1.69 ± 0.64 (s.d.) in homozygotes (1.00 ± 0.15 (s.d.) in wildtypes) but was not significantly different between genotypes (Fig. 2D). All of the Zfp469 transcript expressed in homozygous mutant and 55% ± 3.7% (s.d.) of transcript in heterozygous keratocytes contained the V5 sequence showing that the mutant transcript is expressed at a similar level to the wildtype transcript. This is consistent with a premature stop codon in the single coding exon of Zfp469 resulting in the production of a truncated protein rather than destruction of the transcript by nonsense-mediated decay (NMD) (Fig.2D).

Zfp469 BCS mice recapitulate ophthalmic characteristics of BCS
Clinical characteristics of BCS include thinning of the cornea and sclera, myopia and refractive errors, keratoconus or keratoglobus, corneal rupture and vision loss. We performed ophthalmic phenotyping including slit lamp examination, anterior-segment optical coherence tomography (AS-OCT), histology and immunostaining in order to determine whether the premature stop codon introduced into Zfp469 leads to features of BCS in the mouse. At 3 months of age, by which stage the cornea is fully developed (Hanlon et al., 2011), slit lamp examination revealed no gross corneal opacity or abnormality in homozygous mice (Fig.3A). However, AS-OCT ( Fig.3B) showed that homozygous mice have extremely thin corneas relative to age-and sex-matched wildtype animals, with a mean reduction of 39.1 ± 4.4 µm (s.e.m.) in central corneal thickness (p = 0.0025 in males, p<0.0001 in females, p<0.0001 for sexes combined, one way ANOVA with Tukey's multiple comparison test) (Fig. 3C). This corresponds to a 30% reduction in CCT. Thickness of the peripheral corneal was also significantly reduced by approximately 25% (in female homozygous mice, average 144.6 ± 15.3 µm s.d., n=4; in wildtype females, 187.9 ± 16.2 µm s.d., n=3, p = 0.0335) consistent with generalised thinning of the cornea as is seen in BCS.
Hematoxylin and eosin (H&E) staining to examine corneal morphology confirmed that the multi-layered epithelium appeared normal, as does the intact endothelium (Fig.3D). However, the corneal stroma, which normally comprises 90% thickness of a healthy cornea in humans and approximately 60% of thickness in mice (Hanlon et al., 2011), is markedly thinner in homozygotes.
Extensive shearing was observed between lamellae relative to that seen in wildtype cornea processed for staining in the same way.

Corneal thinning in Zfp469 BCS mice is apparent during corneal development and is not progressive
In some clinical reports, the corneal thinning observed in BCS patients is described as progressive and corresponds with an increasing risk of spontaneous rupture of the cornea or sclera. We sought to determine whether the corneal thinning observed in the Zfp469 BCS/BCS mice at 3 months was established earlier, during development of the cornea, and whether thinning of the stroma was progressive. At 1 month of age, AS-OCT ( Fig.4A) revealed that the central corneas of homozygous animals were, on average, already 46.8 ± 5.6 µm (s.e.m.) thinner than wildtype sex-and agematched controls (Fig.4B, p = 0.0016 males, p = 0.0043 females, p<0.0001 for sexes combined, one way ANOVA with Tukey's multiple comparison test) -a 35% reduction in CCT. Fig.4C shows a unilateral corneal opacity observed in one eye from a Zfp469 BCS/BCS female at 1 month of age (n=12 studied). OCT revealed swelling of the corneal stroma consistent with an edema. It was not possible to determine whether this was a result of injury leading to rupture of Descemet's membrane but there was no evidence of injury or infection. No such accumulation of fluid within the cornea was observed in 26 heterozygous or wildtype eyes at 1 month of age, or in any older mice. Subsequent investigation of corneal thickness at 6 months of age also showed a 30% reduction in CCT, with mean CCT 31.4 ± 5.2 µm (s.e.m.) thinner in Zfp469 BCS/BCS (Fig.4D, E, p = 0.0023 males, p = 0.001 females, p <0.0001 combined). Peripheral corneal thickness was also significantly reduced (in female homozygous mice, average 125.99 ± 14.1 µm, s.d., n=4; in wildtype females, 166.9 ± 21.8 µm s.d., n=3, p = 0.0286). The degree of thinning observed at 1, 3 and 6 months consistently reduced CCT by approximately 30% relative to wildtype age-and sex-matched mice.
The corneas of individual mice with homozygous BCS mutation in Zfp469 -followed from 3 months of age -do not progressively thin up to age 6 months.

Disease Models & Mechanisms • DMM • Accepted manuscript
The thin corneas of homozygous mutant mice aged up to 9 months were not prone to perforation; no animals were affected by corneal damage under normal housing conditions. However, the Zfp469 BCS/BCS corneas were more easily distorted by application of a viscous liquid drop intended to prevent the eye from drying out during OCT than wildtype controls at both 3 and 6 months of age (Fig.4F). We also measured anterior chamber depth (ACD) in AS-OCT images obtained of female mice at 6 months of age. Without dilation of the pupils, ACD in Zfp469 +/+ was 977.1 µm ± 32.9 (mean ± s.d., n=3), in Zfp469 +/BCS 1002.9 µm ± 55.1 (mean ± s.d., n=5), and in Zfp469 BCS/BCS 1016.5 µm ± 32.1 (mean ± s.d., n=4). This difference was not significant but suggested there may be a trend towards increased ACD in eyes of homozygous mutant mice. Subsequently, when the pupils of the same animals were dilated by administration of Tropicamide and Phenylephrine Hydrochloride prior to performing AS-OCT, female Zfp469 BCS/BCS showed a bulging eye phenotype consistent with keratoglobus ( Fig.4G), a condition in which generalised corneal thinning results in globular protrusion of the eye. This was not observed in male mice, or in wildtype females. ACD was determined from OCT images after pupil dilation, confirming a significant increase in ACD in Zfp469 BCS/BCS females relative to wildtype controls at 6 months of age (Fig.4H, Welch's ANOVA test, p=0.0138). Interestingly, the ACD of homozygous mutant eyes decreased by only 2.6% after dilation of the pupil. This contrasts to wildtype ACD which was reduced by 12% on average relative to ACD determined without dilation of the pupil. Together with the specific thinning of the stroma revealed by H&E staining, these data are indicative of a loss of biomechanical strength of the corneal stroma resulting in impaired resistance to both internal and external forces.

Collagen type I, but not type V, is less abundant in the Zfp469 BCS/BCS cornea and fibril architecture is altered
The corneal stroma is a collagenous extracellular matrix generated by resident keratocytes. Having observed thinning of the corneal stroma in Zfp469 BCS/BCS , we next analysed the amount of Collagen I and Collagen V in the corneal stroma. In wildtype corneas at 6 months of age, immunostaining for both collagen I and V showed uniform distribution throughout the stroma, each having a characteristic appearance (Fig.5A). Type I collagen (ColI) staining appears to demarcate linear structures within the stroma; type V collagen staining (ColV) staining had a more punctate appearance within the linear structures. This is consistent with their roles in assembly of collagen fibrils in the cornea, with collagen type I the predominant fibril-forming collagen, whilst collagen type V has an important role in the nucleation of collagen I fibrils. In Zfp469 BCS/BCS cornea sections, the staining of collagen I and V was strikingly confined to a much thinner stroma. Both type I and type V collagen staining in homozygous mutant corneas appeared to be more densely packed than in heterozygous or wildtype corneas but quantitation of fluorescent signals determined that total intensity was not significantly different to wildtype controls (data not shown).
Less Col1a1 Telo collagen was detected by immunoblotting in the lysate of 6 pooled Zfp469 BCS/BCS corneas compared to wildtype, relative to the amount of fibronectin-1 (Fig.5B). These data suggested that collagen production or fibrillogenesis may be affected in BCS corneas, leading us to assess the organisation of the corneal stroma and fibril structure in mice at 9 months of age using transmission electron microscopy (TEM) (Fig.5C). Images obtained from the anterior central cornea showed the lamellae were cylindrical and regularly arranged in both wildtype and mutant corneas, but revealed a striking difference in fibril diameter between 3 wildtype and 3 Zfp469 BCS/BCS samples.
Morphometric analysis revealed that the diameters of cross-sectional collagen fibrils were significantly smaller in Zfp469 BCS/BCS stroma than in wildtype (mean fibril diameter 28.32 nm ± 5.93 nm s.d. compared with 42.53 nm ± 6.32 nm s.d. respectively, p<0.0001). In anterior stroma of individual wildtype and Zfp469 BCS/BCS corneas, there is a narrow distribution of fibril size, as shown in the colour coded images in Fig.5D. Distribution of collagen fibril diameter in 3 corneas per genotype group is shown in Fig.5E. Collagen fibril diameter in the anterior stroma was, on average, 33% reduced in Zfp469 BCS/BCS mice. In addition, an increased number of collagen fibrils were present in a given area of mutant stroma compared to wildtype, with fibril density (fibrils/µm 2 ) of the smaller diameter fibrils being significantly increased from 381.7 ± 47.46 (s.d.) fibrils/µm 2 to 479.3 ± 71.77 (s.d.) fibrils/µm 2 . This corresponds to an increase of 25.6% (Fig. 5F, mutant versus wildtype, p<0.0001) in the stroma of homozygous mutant corneas. Extraocular features of BCS may include generalised connective tissue dysfunction leading to joint hypermobility, easy bruising and soft, doughy skin (Burkitt . During euthanasia, there were 3 instances of tail degloving (separation of the skin and subcutaneous tissues) in Zfp469 BCS/BCS adult mice. No such degloving events occurred for wildtype or heterozygous mice. This suggests that LOF of Zfp469 may compromise the biomechanical strength of the skin or underlying connective tissue. Given that type I collagen is the major component of human skin (85-90%), with type III collagen comprising 8-11% and type V collagen 2-5% of total collagen, we sought to determine whether the skin was affected in Zfp469 BCS/BCS mice. Sections of tail skin from 6 month old mice showed decreased thickness of the dermal and subcutaneous adipose layers in homozygous mutants by H&E staining (Fig.S1A). Masson's Trichrome staining of collagen in the dermis revealed that the skin of Zfp469 BCS/BCS mice contains less collagen than wildtype animals ( Fig.S1). This appears to arise from decreased abundance of type I collagen in the dermis, as shown in a representative image of adult tail skin in Fig. S1C. In keeping with our earlier observation from corneal sections, type V collagen staining did not alter with genotype ( Fig.S1D), suggesting that deficiency of type I collagen underlies both the ocular and extraocular features of BCS.

A primary cell model of stromal composition in Brittle Cornea Syndrome
Keratocytes synthesise components of the healthy stroma and repair tissue damage. There was no significant difference in the number, or density, of stromal keratocytes in Zfp469 BCS/BCS corneal sections visualised by DAPI staining of keratocyte nuclei compared to wildtype controls at 6 months of age (Fig. 5A). In order to determine how loss of function of Zfp469 affects the population of stromal keratocytes, we established primary mouse keratocyte cultures from corneas taken from neonatal pups at p2. At this stage in development of the cornea, keratocytes are still dividing and have yet to become quiescent as in mature corneas. From these cultures of corneal stroma fibroblasts, we investigated the gene expression profile in Zfp469 BCS/BCS primary keratocytes. First, using RT-qPCR we established that the expression of Zfp469 closely reflected that seen in freshly isolated keratocytes, with no significant difference in Zfp469 gene expression between genotypes The proliferative ability of the BCS keratocytes in vitro, determined using the population doubling time for each cell line seeded at the same density at passage 3, was not significantly altered by the loss-of-function mutation in Zfp469 (Fig. 6B). However, 3 out of 4 Zfp469 BCS/BCS primary keratocyte lines did proliferate more slowly than wildtype lines isolated from littermates, suggesting that there may be an impact on the rate of proliferation that we are underpowered to detect. The mean doubling time for Zfp469 +/+ primary keratocytes was 25.86 ± 1.77 (s.e.m.) hours compared to 49.80 ± 13.31 (s.e.m.) hours for Zfp469 BCS/BCS keratocytes (n=4 for each genotype, Fig.6B). Of further note, the 2 mutant cell lines that proliferated most slowly failed to proliferate well beyond passage 4. This was not observed for wildtype cell lines generated from litter mates (data not shown) or for MEFs, which showed no variability in doubling time by genotype (Fig. 6B). This suggests that there might be a genotype, and cell-type, specific effect of Zfp469 mutation on cell proliferation.
The adhesion of keratocytes to ECM is key to establishing and maintaining the structural integrity of the cornea (Parapuram et al., 2011). We sought to determine the impact of LOF of Zpf469 on adhesion of primary keratocytes to a variety of ECM components, including Collagen I, Collagen IV, Fibrinogen, Fibronectin and Laminin. This revealed that Zfp469 BCS/BCS keratocytes adhere less efficiently to fibrinogen (p = 0.0433) and fibronectin (p = 0.0133) compared to wildtype controls ( Fig.6C). Defective adhesion to fibronectin, an ECM protein with known roles in maintenance of tissue architecture and wound healing, may impact upon arrangement and organisation of collagen fibrils in the stroma.
The expression of Prdm5, in which mutations cause BCS type 2, and key components of the stromal ECM was investigated by qPCR (Fig. 6D). Prdm5 expression was unchanged in Zfp469 BCS primary keratocytes. However, a significant impact of Zfp469 BCS allele dosage on the expression of Col1a1 and Col1a2, the genes encoding the alpha 1 and alpha 2 chains of collagen type I that associate to form the triple helix of individual type I collagen, was uncovered. The relative expression of both Col1a1 and Col1a2 was reduced by approximately 50% in homozygous mutant cell lines compared to wildtype lines (Col1a1, adjusted p=0.0001; Col1a2, adjusted p=0.0009, one way ANOVA with Dunnett's multiple comparison test), and by approximately 25% in heterozygous primary keratocytes (Col1a1, adjusted p=0.0014; Col1a2, adjusted p=0.0066) (Fig.6D). The expression of Col5a1 in BCS keratocytes in culture was not significantly altered compared to wildtype cell lines, agreeing with our observations from staining of corneal sections. Expression of Kera, a key keratocyte marker, also showed an allele dosage effect of mutation of Zfp469, increasing 1.5 fold in +/BCS and 3-fold in BCS/BCS keratocyte cell lines (adjusted p=0.0397, one way ANOVA with Dunnett's multiple comparison test). Kera encodes keratocan, a cornea-specific keratan sulfate proteoglycan (KSPG) belonging to the small leucine-rich proteoglycan (SLRP) gene family and it has an important role in regulation of collagen fibril spacing and arrangement in the mature cornea. These data point to a dysfunctional gene expression profile in mutant keratocyte cells that may affect the composition and assembly of the stromal extracellular matrix.
We next investigated the secretion and deposition of type I collagen by primary keratocytes.
The amount of proCol1a1 secreted into the medium by confluent Zfp469 BCS/BCS primary keratocytes maintained in serum-free medium for 7 days was significantly reduced compared to wildtype cells ( Fig.7A, p=0.0332, t-test). Immunoblotting of the same serum-free conditioned medium samples using an anti-Telo Col1a1 antibody confirmed that Zfp469 BCS/BCS primary keratocytes secrete less Col1a1 ( Fig.7B) with densitometry revealing a 43 ± 9 (s.d.) % depletion of Col1a1 secreted relative to wildtype samples. Cell-derived matrices (CDM) from homozygous mutant and wildtype primary keratocytes were generated to study ECM deposition in vitro. Keratocytes were seeded onto gelatincoated plates or coverslips and, once confluent, were treated with ascorbic acid for 20 days in order to induce deposition of a collagen-rich matrix. Immunoblot analysis of the CDMs collected following decellularisation under reducing and denaturing conditions revealed that less type I collagen was deposited by mutant cell lines (Fig. 7C). The expression of the fibronectin-1 gene (Fn1) was unaffected by Zfp469 mutation (Fig. 6D). Using densitometric analysis of Col1a1 Telo signal normalised to fibronectin-1 (Fig. 7D), Zfp469 BCS/BCS primary keratocytes deposited, on average, only 30% of Col1a1 to the CDM relative to wildtype cells (p=0.0097, t-test). Intact, decellularised CDMs were also stained with antibodies against Collagen type I, Collagen type V and Fibronectin (Fig. 7E).
Quantification of fluorescent signals in CDM generated by two Zfp469 BCS/BCS cell lines confirmed that less type I collagen was deposited by Zfp469 BCS/BCS primary keratocytes, whilst collagen type V remained unchanged relative to wildtype (Fig. 7F).

Discussion
Brittle cornea syndrome was first reported in 1968 (Stein et al., 1968) but it was another 40 years until mutations in ZNF469 were identified as a cause of this rare condition. Since then, 30 pathogenic mutations in ZNF469 have been identified, the majority of these resulting in premature stop codons preceding ZF domains. The precise mechanism by which biallelic loss-of-function mutations result in generalised connective tissue dysfunction, and why the most severe manifestation occurs in the cornea and sclera, has remained unclear. This study is the first description of a mouse model of BCS, in which we created a premature stop codon in the orthologous mouse gene Zfp469 to recapitulate the human mutation p.Gly677*. Our results showed that this premature stop codon in the large single coding exon of Zfp469 does not lead to nonsense mediated decay of the mutant transcript and will likely lead to generation of a truncated protein. Despite incorporation of an in-frame V5 tag, we were unable to detect a V5-tagged protein by western blot or immunostaining of tissue samples or fixed primary keratocytes. This suggests that the protein may be expressed at very low levels, the mutated V5 is not recognised or that the V5 tag is inaccessible. Although the mutant protein could not be detected, disrupting Zfp469 clearly perturbs the development of the corneal stroma in mouse, resulting in thinning of the cornea by one third in homozygous mutant eyes at 1 month of age. At 3 and 6 months of age, the mature cornea of Zfp469 BCS/BCS eyes remain 30-35% thinner than wildtypes, suggesting that, in mice, stromal thinning is not progressive.
In humans with BCS, a progressive loss of stromal depth has been described . BCS patients suffer from progressive keratoconus or keratoglobus, with alteration of corneal shape due to distortion observed over a year (Ramappa et al., 2014) or longer (Skalicka et al., 2020). Corneal edema resulting from breaks in the Descemet's membrane may occur, and extremely thin corneas are prone to rupture after minor injury (Burkitt . No longitudinal studies of CCT in BCS have been reported, likely due to the young age at which corneal perforations occur (mean age of published cases with rupture, 4.3 years of age (range 1.5 -19 years (Walkden et al., 2019)). None of our BCS mice, aged up to 9 months, suffered from cornea rupture, and only one case of corneal edema was recorded. This apparent difference in corneal fragility may reflect that the stroma of the mouse cornea contributes only 60% of total thickness compared to 90% in humans, and underlies a proportionally thicker corneal epithelial layer (Hanlon et al., 2011).
The epithelial layer appears unaffected in our model. Despite these anatomical differences, the bulging eye phenotype observed in our BCS mouse model following dilation of the pupil closely resembled keratoglobus. Furthermore, the corneas of homozygous mutant mice showed a decreased ability to withstand corneal strain, deforming after either application of an external eye drop or by dilation of the pupil. This is consistent with a loss of biomechanical strength in the corneal stroma when Zfp469 function is lost, and is in striking contrast to the corneas of wildtype mice, which were able to maintain normal corneal curvature under the same pressure load. This study is the first to show a direct impact of LOF of Zfp469 upon cornea biomechanical strength. However, the genetic association of regulatory variants for ZNF469 with corneal resistance factor (CRF) in genomewide association studies indicates that ZNF469 also plays a role in determining biomechanical properties of the cornea in humans (Jiang et al., 2020;Khawaja et al., 2019;Simcoe et al., 2020).
Numerous studies have shown that disrupting the composition or organisation of the stromal extracellular matrix will affect the shape (Quantock et al., 2003) or strength (Chakravarti et al., 1998) of the cornea. Consistent with this, the corneal thinning caused by loss of Zfp469 appears to result from approximately 50% reduction in the expression of genes encoding type I collagen, Col1a1 and Col1a2, by keratocytes. This is an important result, as the relative abundance of type I and type V collagen in the cornea appears key to the proper organisation of the stroma. Collagen composition in the cornea is unique amongst tissues, with fibrillar collagen in the stroma made up of approximately 80-90% type I and 10-20% type V. Type I collagen assembles into heterotrimers of two alpha 1(I) chains and one alpha 2(I) chain, growing further into heterotypic fibrils with type V collagen. Non-helical terminal extensions of type V collagen protrude from the fibril (Linsenmayer et al., 1993) acting to nucleate fibril growth and determine fibril diameter. Strikingly, the expression of collagen type V remained unchanged in our primary keratocytes cells, in cell derived matrices, and by staining of corneal sections from Zfp469 +/+ and Zfp469 BCS/BCS mice, providing possible insight into the pathomechanisms underlying BCS caused by mutation in Zfp469.
Analysis of fibril diameter, density and organisation in the corneal stroma of our BCS model by TEM further implicated reduced type I collagen expression in corneal stromal thinning. The diameter of collagen fibrils was reduced by 33% in Zfp469 BCS/BCS mice compared to wildtype and fibril density was increased by 25%. This closely resembled the ultrastructural phenotype of the Osteogenesis Imperfecta (OI) mouse model resulting from homozygous mutation in Col1a2 (oim, (Chipman et al., 1993)). CCT of Col1a2 oim/oim corneas is 15% thinner than wildtype controls, and fibril diameter determined by TEM is also reduced by 15%. These features arise from impaired collagen fibrillogenesis as a result of loss of Col1a2 during the assembly of type I collagen heterotrimers (Dimasi et al., 2010). Notably, type I collagen homotrimers containing 3 units of Col1a1 have been observed in adult skin and in OI Pace et al., 2008;Pihlajaniemi et al., 1984;Uitto, 1979). The concurrent decrease in expression of both Col1a1 and Col1a2 may explain the more severe corneal thinning observed in both the BCS model and in BCS compared to OI in humans.
Interestingly, arrangement of fibrils within the lamellae of mature Zfp469 BCS/BCS corneas was regular and the organisation of parallel lamellae appeared normal. Indeed, corneal transparency was maintained throughout our study. A similar observation has been made in a previous report of Col5a1 haploinsufficiency, where heterozygous loss of Col5a1 results in thinning of the mature mouse corneal stroma by 26%, resulting in 14% decrease in total collagen content and a 25% reduction in the number of fibrils in the stroma. Fibril diameter was increased and fibril density was reduced in this model of Ehlers-Danlos Syndrome, but no corneal opacity was observed (Segev et al., 2006). This is in contrast to Col5a1 Δst/Δst mice, where targeted deletion of Col5a1 in the corneal stroma results in total absence of Col5a1 from the stroma, leading to larger and less uniform collagen fibrils and extensive corneal hazing (Sun et al., 2011). These data confirm, firstly, that regular packing of a homogeneous population of fibrils with a narrow distribution of diameters is key to transparency (Hassell and Birk, 2010), and secondly, that the ratio between type I and type V collagen is crucial to the assembly of a well-ordered and mechanically strong corneal stroma.
Despite the evident allele dosage effect upon the expression of both Col1a1 and Col1a2 by keratocytes, with heterozygous cell lines expressing an intermediate amount relative to wildtype and homozygous lines, the corneas of heterozygous Zfp469 BCS mice are only slightly thinner than wildtype. This mimics the observations from heterozygous carriers of LOF ZNF469 and PRDM5 mutations, who appear asymptomatic (Abu et al., 2008;Alazami et al., 2016;Aldahmesh et al., 2012;Avgitidou et al., 2015;Burkitt Wright et al., 2011;Christensen et al., 2010;Khan et al., 2012;Menzel-Severing et al., 2019;Micheal et al., 2016;Ramappa et al., 2014;Skalicka et al., 2020) or show only mild corneal thinning and blue sclera (Burkitt Wright et al., 2011;Khan et al., 2010). This may reflect a critical threshold for the relative abundance of type V collagen to type I collagen in the cornea, which is known to have a major role in determining both the number of fibrils that form and determining the diameter of fibrils (Dimasi et al., 2010;Sun et al., 2011;Wenstrup et al., 2004). Compared to skin, where type V collagen comprises only 2-5% of total fibrillar collagen, the relative ratio of type V to type I collagen in the cornea is normally 5-10 fold higher. In our mouse model of BCS, this relative ratio is increased even further as the amount of type I collagen is reduced in both the corneal stroma and in skin. ZNF469 mutations have been shown to disrupt deposition of type I collagen by dermal fibroblasts (Burkitt Wright et al., 2011), in agreement with our observation of decreased dermal thickness and type I collagen abundance in tail skin from Zfp469 BCS/BCS mice.
However, this is the first report of mutation in Zfp469 disrupting the expression of type I collagen in the corneal stroma, and by primary keratocytes. Consistent with the role of collagen type V as a ratelimiting fibril nucleator, this appears to promote fibril nucleation from the depleted pool of type I collagen synthesised by keratocytes in Zfp469 BCS/BCS corneal stroma. This results in the extremely thin corneal stroma, comprising smaller diameter and more tightly packed collagen fibrils than in wildtype mice, explaining the phenotype observed in our model of the disease and in BCS patients.
Extreme thinning of the cornea was observed at 1 month of age in Zfp469 BCS/BCS mice, and BCS cases have been reported in humans soon after birth (Ramappa et al., 2014) suggesting that this disorder is initiated during development. This has also been observed in a mouse model for Marfan syndrome, another connective tissue disorder, which shows reduced CCT in Fbn1 +/− mice from E16.5 onwards (Feneck et al., 2020). The corneal stroma is established during early development, with collagen deposition by presumptive corneal stromal cells observed at E13 in the mouse (Feneck et al., 2019). As development proceeds, organised collagen fibrils begin to form, directed by keratocyte cell extensions into parallel arrangement. Type I collagen appears to play a key role in this process, as complete knockout of Col1a1 in the Mov13 mouse shows reduced corneal thickness at E16 and structural disorganisation of developing fibrils ( Bard and Kratochwil, 1987). Temporal regulation of There does not appear to be a paucity of keratocytes in the stroma of mature cornea, but we cannot exclude an impact on differentiation from neural crest cells or upon proliferation of keratocytes at early stages of development. Future work using our mouse and cellular models of disease will determine whether corneal thinning in BCS is established during very early stages of development, how ZNF469 regulates expression of type I collagen genes, and if there is a therapeutic window of opportunity for the modulation of ZNF469 function.

Bioinformatics
A literature review was performed to identify mutations in ZNF469 that have been reported in BCS cases. Using the search term "ZNF469", 12 case reports reporting ZNF469 mutations were identified, with a total of 30 different mutations occurring either as compound heterozygotes or homozygous in 53 cases of BCS were implicated. All mutations were mapped to transcript NM_001367624.2 and protein NP_001354553.1 using Ensembl Variant Effect Predictor (VEP, https://www.ensembl.org/Tools/VEP). The Genome Aggregation Database (gnomAD v3.1; https://gnomad.broadinstitute.org/) was searched for BCS mutations that may be present in the general population. Protein sequence alignments were performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). Uniprot (https://www.uniprot.org/) and SMART (http://smart.embl-heidelberg.de/) were used to identify positions of C2H2 zinc finger domains and compositional bias in the amino acid sequence.

Preparation of reagents for genome editing
Paired sgRNA for use with nickase Cas9 were designed using the tool available at Sequences of sgRNA are shown in Table S1. A single-stranded donor oligonucleotide (ssODN) repair template with 5'-56 base pair and 3'-54 base pair homology arms was designed to insert a V5 epitope tag and a premature stop codon into mouse Zfp469 at p.Gly634, and ordered as a PAGE Ultramer DNA Oligo (IDT). After verification of correct sequence by Sanger sequencing, plasmid DNA for each sgRNA was used to test efficiency of on-target editing at the Zfp469 locus in mouse embryonic fibroblasts (MEFs). After selection of the most efficient pair of sgRNA, plasmid DNA was used as a template in PCR to amplify sgRNA 1 and 3 sequences using primers shown in Table S1. DNA was purified using Qiagen PCR purification columns, and 1 µg of purified PCR product was used as template for in vitro transcription using the HiScribe T7 High Yield RNA Synthesis kit (New England Biolabs, E2040S) as described by the manufacturer.

Testing sgRNA in mouse embryonic fibroblasts
Wildtype MEFs (200,000/reaction) were transfected with 2 µg pX458 plasmid containing sgRNA using Neon electroporation (1350V, 30 ms and 1 Pulse). Transfected cells were incubated for 72 hours in a humidified 37°C, 5% CO 2 incubator after which cells were collected. The efficiency of sgRNA-mediated DNA cleavage at the on-target locus was determined using a GeneArt Genomic Cleavage Detection Kit (Invitrogen) as described by the manufacturer. Primers used for target amplicon amplification were Forward 5'-TTCATCTCTGTCACCGCCAT-3' and Reverse 5'-GAAGGGGACAGTCTGGTTGT-3'.

Genome editing in mice
Cytoplasmic injection of 20 µl containing 50 ng/µl SpCas9n mRNA, 50 ng/µl sgRNA, and 150 ng/µl donor oligonucleotides in to C57BL/6J mouse zygotes was performed, followed by immediate transfer to pseudopregnant CD1 females. Founder mice were genotyped by extracting DNA from earclips in DNAreleasy (Anachem) following the manufacturer's instructions. Primers (Forward 5'-TTCATCTCTGTCACCGCCAT-3' and Reverse 5'-GAAGGGGACAGTCTGGTTGT-3') were used to amplify DNA surrounding the editing target site from crude DNA by PCR using DreamTaq Green PCR Master Mix (Thermo Scientific). Sanger sequencing was used to verify editing and repair. One founder mouse with in-frame repair was identified and was used to establish the Zfp469 BCS line by breeding with wildtype (WT, +/+ ) C57BL/6J.

Mouse husbandry
Experiments on mice were performed with UK Home Office project licence approval. Animals were housed in a facility on a 12 hour light/dark cycle with unrestricted access to food and water. All mice were euthanised in accordance to UK Home Office guidelines. Heterozygous F1 offspring of the founder mouse were interbred to generate subsequent generations of Zfp469 BCS mice used in this study, maintained on the C57BL/6J strain background. Litters were genotyped as for the founder mice, or outsourced to Transnetyx (Cordova, TN, USA) using allele-specific custom probes. Male and female mice were used in this study.

Slit lamp examination
The anterior segment of the eyes of 3 Zfp469 +/+ , 3 Zfp469 +/BCS and 3 Zfp469 BCS/BCS mice were examined at 3 months of age using a slit lamp biomicroscope. Mice were examined without anaesthesia, and images taken with a digital camera.

Measurement of eye size
Freshly enucleated eyes from 2 homozygous mice and 3 wildtype mice were placed upon a flat surface, and measured from the front of the cornea to the posterior just left of the nerve four times using calipers. An average measurement was calculated for each eye.

Optical Coherence Tomography
Anterior segment OCT images were captured using a Heidelberg Spectralis OCT. Mice aged 1 month, 3 months and 6 months of age were anaesthetized by inhalation of isoflurane, and corneas were imaged using an anterior segment lens. To test corneal distortion after pupil dilation, a drop of Tropicamide 1% and then a drop of 2.5% Phenylephrine Hydrochloride was added to each eye prior to AS-OCT imaging. Systane Ultra Lubricant (Boots) eye drops were used to prevent eyes drying out.
Corneal dimensions including central and peripheral corneal thickness and anterior chamber depth were determined from cross-sectional corneal images that passed through the centre of the pupil using ImageJ software (National Institutes of Health). CCT and PCT were determined by measuring the linear distance between the anterior and posterior corneal surfaces in the central cornea and peripheral corneal respectively. ACD was the distance between the lens and the posterior surface of the central cornea. The measurements were obtained in pixels and the appropriate pixel to μm conversion factor was applied, relative to 200 µm scale bar. One measurement was made for each scan of both eyes for each mouse. A minimum of 3 male and 3 female mice per genotype were used at each 1, 3 and 6 months of age. GraphPad PRISM were used for data analysis. Imaging and measurements were performed blinded to genotype.

Histology
Mice were euthanised at the appropriate age by cervical dislocation before eyes were enucleated and tails were removed for fixing in in 4% Paraformaldehyde (PFA) solution overnight at 4°C.
Following this, tails were subjected to 3 washes of 6% EDTA for one week each. For wax preservation, tissue samples were removed from PFA or EDTA, and were subsequently dehydrated by successive washes in 70%, 80%, 90% and 100% ethanol, xylene twice and then embedded in paraffin for 45 minutes.
Hematoxylin and Eosin (H&E) and Masson's Trichrome staining were performed on 8 μm paraffin embedded tissue sections using standard procedures. Images were captured on a Zeiss Axioplan 2 brightfield microscope. Immunostaining was performed using 5-8 μm paraffin-embedded sections after antigen retrieval in Citrate Buffer, pH6. Slides were rinsed with water, washed twice with Tris-HCl buffered saline (TBS) with 0.1% Tween-20 in (TBST) and then blocked in 4% heatinactivated donkey serum (DS) in TBST for 30 minutes at room temperature. Primary antibodies (Goat Anti-Type I Collagen, 1310-01, Southern Biotech; Goat Anti-Type V Collagen, 1350-01, Southern Biotech) were diluted 1:100 in 4% DS in TBST and incubated overnight at 4°C. After 3 washes in TBST, Alexa Fluor secondary antibody diluted 1:250 4% DS in TBST was added to the slides for 1.5 hours at room temperature. Slides were washed 3 times in Phosphate Buffered Saline (PBS) prior to mounting coverslips using Prolong Gold Antifade Mountant with DAPI (Invitrogen). Slides were imaged on a Nikon Confocal A1R microscope for image processing using NIS-Elements or ImageJ software.

Corneal protein extraction
For proteomic analysis, 6 pooled corneas (from different 3 animals at 2 months of age) for each genotype were subject to mechanical grinding and 5 rounds of freeze-thawing in RIPA buffer (150 mM NaCl, 50 mM Tris pH 8, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) plus Complete EDTA-free protease inhibitor cocktail (Roche). After centrifugation at 13,000 rpm for 10 minutes at 4°C, supernatants were recovered and protein concentration determined using BioRad Protein assay according to the manufacturer's instructions, with BSA as standard.

Mouse embryonic fibroblast generation and culture
MEFs were isolated from E13.5 embryos from heterozygous Zfp469 +/BCS crosses. Heads and organs were removed from embryos and the remaining sample was digested with trypsin for 10 minutes at 37 °C to generate a cell suspension. Cells were pelleted by centrifugation at 1200 rpm for 4 minutes and then cultured in OPTIMEM + 10% FCS + Pen/Strep. MEFs were used between passages 3-6.

Mouse primary keratocyte generation and culture
Mouse corneal stroma keratocytes were isolated as previously described (Zhang et al., 2016), with slight modification. In brief, pups were euthanised at postnatal day 2, eyes were dissected, rinsed in PBS and corneas cut out along scleral rim. Two corneas (per mouse) were incubated in 15 mg/ml Dispase (4942078001, Roche) in DMEM (Gibco) +Penicillin/Streptomycin for 30 minutes at room temperature. Epithelial cells were detached by gentle shaking and the corneas rinsed in PBS, before incubation in Digestion Buffer containing 2 mg/ml Collagenase (Gibco) and 0.5 mg/ml hyaluronidase (Sigma-Aldrich) in DMEM at 37 °C for 30 minutes, with vortexing every 5 minutes. Samples were centrifuged at 300 x g for 5 minutes before the supernatant was removed, and trypsin/versene added for a further incubation at 37 °C for 10 minutes. Cells were recovered by centrifugation at 300 x g for 5 minutes, supernatant discarded and cells resuspended in culture medium (DMEM, 10% Fetal Calf Serum (FCS), 1% Pen/Strep) for plating. Primary keratocytes (corneal stromal fibroblasts) were maintained in a humidified 37°C, 5% CO 2 incubator, sub-cultured at 80% confluency, and were used between passage 3-6 during this study.

Isolation of RNA, cDNA synthesis and real-time quantitative PCR (qPCR)
RNA was isolated from corneal samples dissected from 4 wildtype, 2 heterozygous and 4 homozygous mice aged 3 months and the corneas processed as described to obtain cells for primary keratocytes culture. After treatment with trypsin/versene, the resulting cell pellet was resuspended in Buffer RLT and total RNA prepared using an RNEasy Kit (Qiagen). RNA was also purified from primary mouse keratocytes isolated from p2 pups at passage 4 using the RNEasy kit. Reverse transcription was performed using the Transcriptor High Fidelity cDNA synthesis kit (Roche) and random hexamer primer according to manufacturer's instructions. qPCR was performed using Taqman Gene Expression assays (Hprt, Mm01324427_M1, Col1a1, Mm00801666_g1, Col1a2, Mm00483888_m1, Col5a1, Mm00489229_m1;Kera, Mm00515230;Fn1, Mm01256744_m1;Prdm5, Mm00510567_m1;Gapdh, Mm99999915_g1; custom assays targeting start of the Zfp469 transcript, and the V5 insertion; Applied Biosystems) with Taqman Universal MasterMix II, no UNG (Applied Biosystems). Assays were run on an ABI PRISM 7900 thermocycler with each sample in triplicate.
Hprt and Gapdh were used as housekeeping genes, and data were analysed using the 2 -ΔΔCT method for relative quantitation.

Proliferation assays
Proliferation assays were performed using 50,000 primary mouse keratocytes or MEF from four independent cell lines for each genotype at passage 3. After 96 hours in culture complete culture medium the cells were trypsinised and counted using a haemocytometer. The number of population doublings in 96 hours was used to calculate the doubling time for each cell line in hours.

Adhesion assay
The adhesion of primary mouse keratocytes to the ECM components Collagen I, Collagen IV, Fibrinogen, Fibronectin and Laminin was quantitatively tested using a CytoSelect Adhesion Assay Kit (Cell Biolabs, San Diego, CA) as described by the manufacturer. In brief, three wildtype lines, obtained from three different mice, were compared to three homozygous mutant lines, also obtained from three different mice. 75,000 cells in serum-free DMEM were seeded into wells of a 48-well plate pre-coated with the ECM components and incubated for 45 minutes at 37°C. Media was removed and the plate washed 5 times with PBS to remove non-adherent cells, before addition of Cell Stain Solution to each well. After incubation for 10 minutes at room temperature, wells were washed 5 times with PBS and allowed to air dry. Extraction Solution was added and after 10 minutes incubation with shaking, 150 µl of each sample was used to measure absorbance at 560 nm in a TECAN M200 Pro plate reader (TECAN, Switzerland).

Pro-Collagen 1A1 Enzyme-linked Immunosorbent Assay (ELISA)
Primary mouse keratocytes were seeded 75,000 cells per 10 cm plate and grown to confluency in medium containing FCS. Cells were washed in PBS, and medium changed to serum-free for a further 7 days. Cell culture supernatants were collected and assayed for the secretion of pro-Col1A1 using Human Pro-Collagen I alpha 1 DuoSet ELISA kit (DY6220-05, R&D Systems) as described by the manufacturer.

Generation of Cell-Derived Matrices (CDM)
Primary mouse keratocytes cells were used to generate CDMs following a published protocol (Kaukonen et al., 2017). In brief, multi-well tissue culture plates and glass coverslips were coated with sterile 0.2% (w/v) gelatin diluted in PBS for 1 hour at 37°C. Coverslips were washed with PBS and then cross-linked with 1% (v/v) glutaraldehyde for 30 minutes at room temperature, prior to quenching with sterile 1 M glycine for 20 minutes. Coverslips and plates were then washed with PBS and incubated in DMEM, 10% FCS, 1% Pen/Strep for 1 hour before use.
Media was then supplemented with 50 μg/ml ascorbic acid with complete media changes every 2 days for 20 days. After washing in PBS, cells were denuded by adding Extraction Solution (20 mM NH 4 OH, 0.5% Triton X-100 in PBS) to lyse the cells. Following two washes in PBS, DNA was digested with 10 μg/ml DNase I (Roche) in PBS for 30 minutes at 37°C, before washing twice more. CDMs in tissue culture plates were scraped into 2 x LDS Buffer with reducing agent added before denaturing at 90°C for 20 minutes. CDMs on coverslips were fixed in 10% formalin for 15 minutes at room temperature, washed with PBS and blocked in 30% DS in PBS. Primary antibodies (1:250 Anti-Fibronectin-1, F3648, Sigma-Aldrich; 1:100 Goat Anti-Type I Collagen, 1310-01, Southern Biotech; 1:100 Goat Anti-Type V Collagen, 1350-01, Southern Biotech) were diluted in 30% DS in PBS and incubated on CDMs for 1 hour at room temperature. After 3 washes in PBS, Alexa Fluor conjugated secondary antibodies (Donkey Anti-Goat Alexa Fluor 488, A-11055, Thermo Fisher Scientific; Donkey Anti-Rabbit Alexa Fluor 594, A21207, Thermo Fisher Scientific) were added for 1 hour at room temperature. Finally, 3 washes in PBS and one in water preceded mounting of the coverslips on slides using Vectashield Antifade mounting medium (Vector Laboratories). Imaging was performed using a Nikon Confocal A1R microscope. NIS-Elements or ImageJ software was used for image processing and analysis.

Statistics
Statistically significant differences between experiments were determined using unpaired Student's T-test (two-tailed), one-way ANOVA or Welch's ANOVA test where variances between groups differed significantly. Post hoc analysis using Tukey's or Dunnett's multiple comparisons tests was performed (GraphPad Prism v9.1.0). A p-value of less than 0.05 was considered significant.
Experiments were performed a minimum of 3 times, unless indicated otherwise, and data is presented as mean ± standard error of the mean (s.e.m.) or ± standard deviation (s.d.).      Cell-derived matrices (CDM) generated by primary keratocytes were decellularised and then denatured and reduced prior to western blotting. The CDM of Zfp469 BCS/BCS contained less Telo Col1a1 than wildtype CDM, using fibronectin as a loading control. (D) Quantification of Col1a1 signal normalised to fibronectin for CDMs generated from 3 Zfp469 +/+ and 3 Zfp469 BCS/BCS by densitometry shows a significant decrease in abundance of Telo Col1a1 in mutant cell lines (n=3 per group, p= 0.0097, unpaired two-tailed t test). (E) Representative immunofluorescence images of CDMs generated by primary keratocytes after 20 days exposure to ascorbic acid-containing medium, stained for type I collagen (green), type V collagen (green) and fibronectin (red). Scale bar represents