Purpose: Brittle Cornea Syndrome (BCS) is a rare recessive condition characterized by extreme thinning of the cornea. The collagen-rich corneal stroma accounts for 90% of corneal thickness in humans, and is a major determinant of visual acuity. BCS results from homozygous loss-of-function mutations in ZNF469 (BCS1) or PRDM5 (BCS2). Our mouse model of BCS1, caused by a premature termination codon (PTC) mutation in Zfp469, showed that the stroma is thinner due to of decreased type 1 collagen expression 1 . To elucidate disease mechanisms and test potential therapies, we established a primary keratocyte disease-in-a-dish model of BCS1. Translational readthrough inducing drugs (TRIDs) can restore full-length proteins truncated by PTC and are a potential treatment for 20% of BCS patients with PTC mutations. Primary keratocytes from our BCS1 mouse model were treated with TRIDs to assess their efficacy in improving the BCS phenotype in vitro. Methods: Zfp469 BCS1/BCS1 keratocytes were treated with TRIDs PTC124 (ataluren), amlexanox, gentamicin, geneticin or vehicle-only, for 2 or 5 days. Following treatment, Zfp469 and Col1a1 mRNA levels were evaluated by RT-qPCR. Changes to keratocyte cell-derived matrices were assessed using immunostaining or the collagen dye CNA35. Results: Zfp469 BCS1/BCS1 keratocytes express 40% more Zfp469 mRNA than wildtype cells; this was unchanged by treatment with amlexanox, geneticin or gentamicin. PTC124 decreased expression of Zfp469 by 50%. Expression of Col1a1 , a key component of the stromal matrix, was significantly decreased by PTC124 treatment (<65% relative to vehicle-only Zfp469 BCS1/BCS1 keratocytes). Collagen deposition, visualized using CNA35 dyes, was decreased by PTC124 treatment in BCS and wildtype keratocytes. Conclusions: BCS is a devastating condition. Extreme thinning of the corneal stroma, as a result of impaired collagen production by keratocytes, makes the cornea prone to rupture. This causes irreversible sight loss. There is currently no treatment for BCS. Our initial work suggests that TRIDs may not restore functional full-length Zfp469 in primary keratocytes and are unlikely to be of therapeutic benefit in BCS. Reference 1. Stanton CM et al. A mouse model of brittle cornea syndrome caused by mutation in Zfp469. Dis Model Mech. 2021;14(9):dmm049175.

Purpose: Following genome-wide association study for risk factors for retinal detachment, a locus overlapping the BMP3 gene, previously reported to be associated with myopia, was identified and selected for further examination. We prioritized an associated missense plausibly deleterious variant within the BMP3 gene for functional analysis. The BMP signalling pathway has long been implicated in the patterning and development of the eye, but BMP3's role both within this pathway and during eye development and disease is unclear. Methods: The region around the associated missense variant is conserved between human and mouse, and the missense variation was introduced using CRISPR-cas9 genome editing; loss of function mutations were also produced in the process. Our unit is uniquely equipped to analyse mouse eye phenotypes and using Optical Coherence Tomography (OCT), electroretinogram (ERG) and Optokinetic Drum (OKD) these mice were comprehensively phenotyped through to adulthood. RNA sequencing analysis was also used in cell lines and in primary mouse tissue to further investigate disrupted signalling pathways and potential downstream targets of Bmp3. Results: OCT of mice showed no gross morphological abnormalities in either mice carrying the missense variant or loss of function mutations. ERG and OKD also showed that retinal function and visual acuity was unaltered in either line. However axial length measurements of adult mice showed a significant increase in length of the eye – indicative of myopia. Transcriptional analysis of two different retinal pigmented epithelial cell lines carrying loss of function mutations have been compared to results obtained from whole eye cups of mutant mice, and we have highlighted potential differences and similarities between the two. Conclusions: Future work is required to assess how much the mouse and human cell line results converge, but our mouse results support a role for a BMP pathway in myopia development.

Purpose: Brittle Cornea Syndrome (BCS) is a rare disease that affects 1 in 1 000 000 people world wide. With a reduction in cornea thickness and therefore strength sufferers have an increase in loss of vision through injury. Our lab has previously published the first mouse model of BCS Type 1 (1) and are currently characterizing a novel model of BCS type 2 in order to elucidate the underlying mechanisms causing this disease. Methods: Mice carrying a 7 bp deletion which causes a frameshift and premature stop codon within the causative gene Prdm5 have been extensively phenotyped using Optical Coherence Tomography (OCT), Transmission Electron Microscopy (TEM) and Histology. Results: These mice show a reduced central corneal thickness by OCT from as early as 1 month of age. TEM and histological analysis indicates that this reduction in thickness may be due to the dysregulation of collagen assembly – as stromal collagen fibre diameter is significantly reduced. Conclusions: The structure of the stroma is crucial to corneal function: mechanical strength and transparency are achieved by the intricate arrangement of collagen fibrils, synthesized by keratocytes. Although it is known that the corneal stroma has a unique composition in which the ratio of Type I to Type V collagen is very different to anywhere else in the body, it remains unclear how the synthesis and maintenance of this specialized extracellular matrix by keratocytes is controlled. Characterization of the nature and extent of developmental defects in the cornea of this mouse model will be important to devise future therapeutic interventions. Reference 1. Stanton, C. M., Findlay, A. S., Drake, C., Mustafa, M. Z., Gautier, P., McKie, L., Jackson, I. J., & Vitart, V. (2021). A mouse model of brittle cornea syndrome caused by mutation in Zfp469. Disease models & mechanisms, 14(9), dmm049175. https://doi.org/10.1242/dmm.049175

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping early-career researchers promote themselves alongside their papers. Chloe Stanton is first author on ‘ A mouse model of brittle cornea syndrome caused by mutation in Zfp469’, published in DMM. Chloe is a postdoc in the lab of Dr Veronique Vitart at The University of Edinburgh, Edinburgh, UK, investigating the genetic and molecular mechanisms underlying eye diseases.

Late-onset retinal degeneration (L-ORD) is an autosomal dominant macular degeneration characterized by the formation of sub-retinal pigment epithelium (RPE) deposits and neuroretinal atrophy. L-ORD results from mutations in the C1q-tumor necrosis factor-5 protein (CTRP5), encoded by the CTRP5/C1QTNF5 gene. To understand the mechanism underlying L-ORD pathology, we used a human cDNA library yeast two-hybrid screen to identify interacting partners of CTRP5. Additionally, we analyzed the Bruch's membrane/choroid (BM-Ch) from wild-type (Wt), heterozygous S163R Ctrp5 mutation knock-in (Ctrp5(S163R/wt)), and homozygous knock-in (Ctrp5(S163R/S163R)) mice using mass spectrometry. Both approaches showed an association between CTRP5 and HTRA1 via its C-terminal PDZ-binding motif, stimulation of the HTRA1 protease activity by CTRP5, and CTRP5 serving as an HTRA1 substrate. The S163R-CTRP5 protein also binds to HTRA1 but is resistant to HTRA1-mediated cleavage. Immunohistochemistry and proteomic analysis showed significant accumulation of CTRP5 and HTRA1 in BM-Ch of Ctrp5(S163R/S163R) and Ctrp5(S163R/wt) mice compared with Wt. Additional extracellular matrix (ECM) components that are HTRA1 substrates also accumulated in these mice. These results implicate HTRA1 and its interaction with CTRP5 in L-ORD pathology.