Frank Rutsch (1), Yvonne Nitschke (1), Joseph Deering (2), Dominique Bazin (3), Christoph R. Clemens (4), Uwe Hansen (5), Nicole Eter (4), Marc D. McKee (2,6), Peter Heiduschka (4)
Affiliation(s):
1. Department of General Pediatrics, Muenster University Children’s Hospital, Albert-Schweitzer Campus 1, Gbde. A1, D-48149 Münster, Germany;
2. Faculty of Dental Medicine and Oral Health Sciences, McGill University, Montreal, Canada
3. Laboratoire de Physique des Solides, CNRS, Université Paris-Sud, 91405 Orsay, France
4. Department of Ophthalmology, Muenster University Hospital, Münster, Germany
5. Institute for Musculoskeletal Medicine, Muenster University Hospital, Münster, Germany
6. Department of Anatomy and Cell Biology Faculty of Medicine and Health Sciences, School of Biomedical Sciences, McGill University, Montreal, Canada
Objectives: Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is the most important generator of extracellular inorganic pyrophosphate, an inhibitor of matrix mineralization. Variants in the ENPP1 gene lead to calcifications in the media of arteries and intimal proliferation. In ENPP1 deficiency, retinal changes have not yet been thoroughly investigated.
Here we report on the ophthalmological status of a patient suffering from ENPP1 deficiency, and we also characterize the eyes of ttw/ttw mice carrying the homozygous Enpp1 variant p.Gly568* associated with ectopic calcifications.
Methods: The patient (male, 27 years) is a homozygous carrier of the ENPP1 variant p.Glu893*. He was examined ophthalmologically over a duration of seven years, including infrared scanning laser ophthalmoscopy (SLO), spectral domain optical coherence tomography (SD-OCT), OCT angiography and electroretinography (ERG). Observations were compared with age-matched healthy controls.
The ttw/ttw mice received a high-phosphate diet to accelerate calcification. Their eyes were studied at an age of 12 weeks by OCT and fluorescein angiography (FAG), and retinal function was assessed by ERG. Observations were compared with wild-type mice. Enucleated eyes were studied by alizarin red, von Kossa and OsteoSense staining, by submicron X-ray microscopy, transmission electron microscopy and scanning electron microscopy. Calcium content of the mouse eyes was determined by the O-cresolphthalein-complexon method. Calcium deposits were additionally examined by X-ray diffraction, energy-dispersive spectroscopy and optical photothermal infrared spectroscopy.
Results: In the patient, numerous small yellowish dots were seen in the middle and peripheral regions of the retina, similar to a fundus albipunctatus. They appeared hyperreflective by infrared SLO, did not change during the seven years of observation, and did not have a correlate in SD-OCT images. The retina and retinal nerve fibre layer (RNFL) at the optic disc were thinner in the nasal direction than in healthy controls (retina 348 µm vs. 373 µm, p=0.0009, RNFL 80.5 µm vs. 131.5 µm, p=0.0003). By OCT angiography, vessel densities were at the lower limit of the normal range. ERG revealed normal latencies, but decreased amplitudes compared to healthy controls (between 50 and 90% of control values).
The eyes of ttw/ttw mice showed a normal appearance by OCT and FAG. Retinal layers had a similar thickness as in control mice. Nodular calcific deposits measuring tens of micrometers were found localized to the region of Bruch’s membrane (BM) and the retinal pigment epithelium along the ciliary margin of the retina, with flattened calcifications observed in an aligned arc of deposits extending towards the back of the eye. The deposits contained amorphous calcium phosphate and hydroxyapatite. Occasionally, electron dense structures were associated with collagen fibrils in the BM. ERG amplitudes were smaller than in control animals (at ~80%).
Significance: Morphological changes of the retina in human ENPP1 deficiency are subtle but can be associated with retinal impairment. This may reflect decreased photoreceptor function caused by reduced nutrient supply to the photoreceptors due to calcification of the BM and adjacent tissue. ENPP1-deficient ttw/ttw mice presenting with calcific retinal deposits recapitulate functional retinal impairment and serve as a model for age-related macular degeneration and related disorders associated with retinal calcifications.