June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
A cellular model for Brittle Cornea Syndrome
Author Affiliations & Notes
  • Chloe Stanton
    The University of Edinburgh MRC Human Genetics Unit, Edinburgh, Edinburgh, United Kingdom
  • Camilla Drake
    The University of Edinburgh MRC Human Genetics Unit, Edinburgh, Edinburgh, United Kingdom
  • Amy Findlay
    The University of Edinburgh MRC Human Genetics Unit, Edinburgh, Edinburgh, United Kingdom
  • Zuhair Mustafa
    The University of Edinburgh MRC Human Genetics Unit, Edinburgh, Edinburgh, United Kingdom
  • Susan Campbell
    The University of Edinburgh MRC Human Genetics Unit, Edinburgh, Edinburgh, United Kingdom
  • Veronique Vitart
    The University of Edinburgh MRC Human Genetics Unit, Edinburgh, Edinburgh, United Kingdom
  • Footnotes
    Commercial Relationships   Chloe Stanton, None; Camilla Drake, None; Amy Findlay, None; Zuhair Mustafa, None; Susan Campbell, None; Veronique Vitart, None
  • Footnotes
    Support   Carnegie Trust Research Incentive Grant RIG009328
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 762. doi:
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      Chloe Stanton, Camilla Drake, Amy Findlay, Zuhair Mustafa, Susan Campbell, Veronique Vitart; A cellular model for Brittle Cornea Syndrome. Invest. Ophthalmol. Vis. Sci. 2021;62(8):762.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : The corneal stroma accounts for 90% of corneal thickness in humans, and is a major determinant of visual acuity. The rare recessive condition Brittle Cornea Syndrome (BCS), characterised by extreme thinning of the cornea and sclera and general connective tissue dysfunction, results from loss of function mutations in the poorly understood genes ZNF469 or PRDM5. PRDM5 (PR/SET Domain 5), a widely expressed transcription factor, plays an important role in extracellular matrix (ECM) production by skin fibroblasts and in bone. It is an excellent candidate to regulate ECM development and maintenance in keratocytes, the cells that synthesise the corneal stroma. Using an in vitro model of PRDM5 deficiency we aimed to elucidate the mechanisms by which PRDM5 maintains the transcriptional and proteomic profile of keratocytes to influence stromal thickness in health and disease.

Methods : CRISPR-Cas9 genome editing was used to recapitulate human BCS mutations in PRDM5 in a human keratocyte cell line, hTK. Cell lines were subject to transcriptomic profiling, and the impact of PRDM5 mutation upon the composition of cell-derived matrices assessed using western blotting, immunostaining and mass spectrometry.

Results : Homozygous frameshift mutations in PRDM5 created using CRISPR-Cas9 genome editing resulted in premature termination codons preceding all 16 C2H2 zinc finger domains, mimicking the consequences of pathogenic mutations identified in BCS patients. Expression of PRDM5 was reduced in the edited cell lines, and PRDM5 protein was not detected in cell lysates, consistent with nonsense mediated decay of mutant transcripts. The expression of genes with important roles in keratocytes was altered by loss of function of the transcription factor PRDM5, changing the composition of ECM in vitro.

Conclusions : The creation of a cellular model of PRDM5 dysfunction in a keratocyte cell line offers a unique entry point for investigating the poorly understood regulatory processes shaping the stroma in health and disease. Transcriptomic and proteomic analyses highlight pathways that contribute to the development and maintenance of a healthy corneal stroma. Work remains to determine whether these pathways may be modulated for diagnostic or therapeutic benefit in conditions such as BCS or keratoconus where the cornea thins progressively over time. Furthermore, the mechanisms uncovered by this study may have wider implications in connective tissue disorders.

This is a 2021 ARVO Annual Meeting abstract.

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