Correlation of clinical fundus images with molecular and histopathological analyses of calcified Bruch’s membrane in patients with pseudoxanthoma elasticum

Abstract

Purpose : Pseudoxanthoma elasticum (PXE) is a rare genetic disorder leading to ectopic calcification of Bruch’s membrane (BrM). This can result in a reduced metabolic exchange between the retina and choroid, atrophy of retinal pigment epithelium, and formation of angioid streaks with choroidal neovascularization, all of which can lead to vision loss. Here, we present a histopathological study to investigate the molecular composition and distribution of calcified BrM in eyes with PXE, followed by correlation with clinical images.

Methods : Six cadaveric eyes that were clinically diagnosed with PXE were obtained from the Netherlands Institute for Neuroscience, Amsterdam or the Dutch National Expertise Center for PXE, Utrecht. Eyes were either flat-mounted on to glass slides or embedded in paraffin wax and sectioned to 7 µm. To visualize the distribution of calcified BrM, flat-mounted and cross-sectioned tissues were stained with OsteoSense 680EX, a fluorescent dye specific for the calcium phosphate, hydroxyapatite. The elemental composition of BrM was analysed using energy dispersive x-ray spectroscopy (EDX) whilst time of flight-secondary ion mass spectrometry (TOF-SIMS) was used for mineral composition analysis.

Results : In PXE eyes, the distribution of BrM calcification changes across the central-peripheral axis. On flat-mounted eyes, extensive OsteoSense labelling was observed in the central region with a marked reduction and a patchy distribution towards the periphery. On cross-sections, OsteoSense labelling confirmed the presence of a confluent layer of calcification in the central region with intermittent staining in peripheral regions. EDX analysis confirmed the enrichment of calcium and phosphorus in the BrM of PXE eyes and showed a distribution similar to OsteoSense staining. TOF-SIMS confirmed that some calcifications, at least, were formed of hydroxyapatite.

Conclusions : Extensive calcification is present in the BrM of eyes from PXE patients. Correlation of histopathology and molecular analyses with clinical images suggests that the phenotype observed on fundus imaging modalities is likely associated with the distribution of calcification in the BrM. For example, confluent calcification is associated with angioid streaks whilst the patchy calcification observed in the periphery is likely associated with regions of p'eau d'orange.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.