1. Correlation of in-vivo confocal microscopy findings with clinical severity of aniridia related keratopathy. Mandal N 1 Shortt A.J. 1,2,3 1. Cornea and External Disease Service, Moorfields Eye Hospital NHS Foundation Trust, London UK 2. Department of Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, London UK 3. NIHR Biomedical Research Centre, Moorfields Eye Hospital, London

2. No financial interests to declare

3. Purpose Congenital aniridia is a bilateral inherited eye disease caused by a mutation in one of the PAX6 gene alleles, which in turn leads to low levels of the PAX6 transcription factors Aniridia related keratopathy (ARK) occurs in 90% of patients Is limbal epithelial stem cell (LESC) deficiency the pathogenesis behind ARK or rather secondary to corneal epithelial-keratocyte cell interactions The aim of this study was to determine whether the clinical signs of ARK correlate with changes in the corneal epithelium and anterior stromal keratocyte cell populations and the sub-epithelial nerve plexus observed using in vivo confocal microscopy (IVCM)

4. Methods 3 normal subjects and 8 patients with ARK of varying degrees of severity. The photographs were graded using the Clinical Outcome Assessment in Surgical Trials of Limbal stem cell deficiency (COASTL) tool developed by our group Grading of ARK severity based on: 1) Corneal epithelial haze. 2) Superficial corneal neovascularisation. 3) Corneal epithelial irregularity. Each graded from 0 (normal) to 3 (severe). Then summated to give a score of overall severity IVCM: calculate mean corneal basal epithelial cell density (cells/mm 2 ) and anterior stromal keratocyte cell density (cells/mm 2 ) IVCM : describe morphology and distribution 3 1 2

5. The mean (±SD) scores for aniridia patients and normal controls were 4.27±2.3 (n=8) and 0 (n=3) respectively (p<0.05). Score range: 3 to 9. Control patients all had a grading score of 0. Results

6. Morphology: Increasing disease severity A-C. 1)Basal cells. 2)Keratocytes. 3)Nerve plexus 3 2 1

7. Discussion This study adds to our understanding of the way in which the Pax6 mutation effects the density, distribution and morphology of the individual cell types Our data supports previous findings Suggests ARK maybe primarily due to an abnormal corneal epithelial/ stromal healing response, alterations in the microenvironment and/or abnormal differentiation of progenitor cells Implications for therapeutic targets? Future analysis: IVCM analysis of clinically healthy looking corneas of aniridia patient patients to determine any cellular changes that precede clinical manifestation.

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