Patient Characteristics

Diagnosis of PXE was confirmed by (1) genetic testing using direct sequencing and multiplex ligation-dependent probe amplification analysis of the ABCC6 gene, as described previously or (2) skin histopathology in combination with characteristic funduscopic findings, or both. All patients underwent a complete ophthalmologic examination, including best corrected visual acuity, funduscopy, fundus photography, confocal laser scanning ophthalmoscopy, optical coherence tomography (OCT) and, in selected cases, fluorescein or indocyanine green angiography or both. Refractive error (spherical equivalent) was measured by autorefractometry (NIDEK ARK-560A; NIDEK, Gamagori, Japan).

Patients with any additional retinal pathology known to affect choroidal thickness, prior retinal surgery or any other previous treatment, including laser photocoagulation or photodynamic therapy, were excluded. Use of intravitreal vascular endothelial growth factor inhibitors was not an exclusion criterion. Subjects with refractive errors higher than ± 3 diopters spherical equivalent were excluded from analysis.

Eyes of patients with PXE were grouped according to certain disease characteristics, assuming more severe Bruch membrane alterations with more progressed disease stages ( Figure 1 ). Group 1 included eyes with characteristic PXE-associated fundus changes, such as angioid streaks, peau d’orange and peripheral comet lesions without evidence of CNV or macular chorioretinal atrophy ( Figure 1 , left). Group 2 was characterized by active CNV or secondary subretinal fibrosis ( Figure 1 , middle). Group 3 included eyes with chorioretinal atrophy without evidence for CNV ( Figure 1 , right). Presence or absence of CNV was confirmed using fluorescein angiography and OCT imaging. If applicable, the right eye of each patient was assigned to 1 of the 3 groups. If the right eye matched 1 of the exclusion criteria, the left eye was used for analysis. If patients had a different phenotype in each eye, both could be included and were assigned to different groups.

Characteristics of the 3 different subgroups of pseudoxanthoma elasticum (PXE)-related fundus changes. Patients with PXE were subdivided into 3 groups based on clinical findings. Group 1 included subjects with characteristic PXE-associated fundus changes in the absence of choroidal neovascularization (CNV) or chorioretinal atrophy (left); Group 2 included patients with additional CNV (middle); and Group 3 included patients with progressive chorioretinal atrophy without CNV activity (right).

The right eyes of control subjects without any ocular disease served as controls. Controls underwent an ophthalmologic examination including best corrected visual acuity, funduscopy and fundus photography.

Image Acquisition and Processing

Enhanced depth imaging (EDI) OCT was performed using the enhanced-depth imaging mode of a spectral domain OCT device (Spectralis HRA-OCT, Heidelberg Engineering, Heidelberg, Germany). Single horizontal and vertical line scans were recorded centered on the fovea with up to 100 images averaged. Localization on the foveal center was controlled manually.

Choroidal thickness was measured manually using the Heidelberg Eye Explorer software (Heidelberg Engineering). Thickness was measured from the outer border of the highly reflective RPE/Bruch membrane band to the sclerochoroidal junction as described by Margolis and Spaide. Measurement was performed by 2 independent readers (MG and PLM) subfoveally and in 500-μm intervals up to 3 mm nasal, temporal, superior, and inferior to the foveal center, as exemplified in a horizontal OCT scan in Figure 2 . For en face images, volume scans encompassing an area of 15 degrees × 10 degrees with 97 scans (distance between scans ∼30 μm) and 15 images averaged per scan were acquired. Images were generated using a prototype transverse section viewer tool from Heidelberg Eye Explorer software. Bruch membrane was automatically identified in all sections by the software and served as a reference plane. If needed, the reference plane was adjusted manually. Choroidal en face images were then computed based on the information of the volume scan as a single layer 60 μm beyond the reference plane, based on previous experience using this algorithm.

Measurement of choroidal thickness in enhanced-depth imaging optical coherence tomography (EDI-OCT) images of a representative healthy control without pseudoxanthoma elasticum. Choroidal thickness was measured from the hyperreflective retinal pigment epithelium band to the sclerochoroidal junction in horizontal and vertical EDI-OCT scans through the foveal center. Thickness was measured subfoveally and in 500 μm intervals up to an eccentricity of 3000 μm.

Statistical Analysis

Statistical analysis was performed using commercially available statistic software (IBM SPSS, v 20.0, IBM, Armonk, New York, USA). Mean subfoveal choroidal thickness was compared among the groups by one factorial analysis of variance (ANOVA) followed by the Tukey test for the pair-wise comparison of groups. To assess the difference in the age dependency of subfoveal choroidal thickness between patients with PXE compared to controls, we fitted a linear model to the logarithmic transformed thickness values. Assuming that the early average thickness was the same in both groups, we chose a model with identical intercept but different slopes. Correlation between refractive error and choroidal thickness was assessed by computing the Pearson correlation coefficient. Interobserver agreement was determined by calculating the intraclass correlation (ICC) coefficient for subfoveal choroidal thickness (ICC r = 0.98). There were 10 patients in whom both eyes were included in the study. In these cases, both eyes belonged to different subgroups of the disease, so we assumed the effect of the dependency between the two eyes of one person to be negligible for our analyses.

Subfoveal Choroidal Thickness in Pseudoxanthoma Elasticum

Subfoveal choroidal thickness was significantly different across the 4 groups (ANOVA P < 0.001). Compared to controls, mean subfoveal choroidal thickness was thinner in patients with PXE overall and in all 3 subgroups ( P < 0.001 for each group) ( Table , Figure 3 ). Comparison between PXE subgroups revealed thinner subfoveal choroidal thicknesses in groups 2 and 3 compared to Group 1, and a further reduction in Group 3 compared to Group 2 ( P = 0.037, P < 0.001, P = 0.015) ( Table , Figure 3 ). Group 1 included 2 patients with subretinal fluid in absence of a CNV. The respective eyes showed choroidal thinning similar to the other patients in Group 1.

Subfoveal choroidal thickness in pseudoxanthoma elasticum (PXE). The box plots show measurements of subfoveal choroidal thickness in controls and 3 subgroups of PXE-related fundus changes (see Figure 1 ). Patients with PXE showed a significantly thinner choroid compared to controls. Between the PXE subgroups, there was progressive thinning from Group 1 (no choroidal neovascularization (CNV) or chorioretinal atrophy) to Group 3 (chorioretinal atrophy), with an intermediate effect in Group 2 (CNV). * = P < 0.001

Topography of Pseudoxanthoma Elasticum–Related Choroidal Thickness Changes

To further study the topography of choroidal changes, choroidal thickness was measured at various locations in 500 μm intervals up to an eccentricity of 3000 μm nasally, temporally, superiorly, and inferiorly ( Figure 4 ).

Topography of choroidal thickness in pseudoxanthoma elasticum (PXE). The graphs show the topographic distribution of choroidal thickness along horizontal (top row) and vertical (bottom row) enhanced-depth imaging optical coherence tomography (EDI-OCT) line scans. The graphs on the left represent the mean at different measurement positions along EDI-OCT scans; the graphs on the right show the ratio of mean values of PXE subgroups to controls. Error bars and the gray area show 95% confidence intervals. All 3 PXE subgroups showed an overall reduced choroidal thickness compared to controls. Group 1 exhibited the least, and Group 3 the largest reduction of choroidal thickness. Choroidal thinning was most pronounced close to the optic disc and approximated normal control values with increasing distance from the optic disc.

The choroid in control eyes was thickest subfoveally and showed a continuous thinning with increasing eccentricity along horizontal and vertical scans through the foveal center ( Figure 4 , left). The thinnest values of choroidal thickness were measured 3000 μm nasally.

In all 3 subgroups of the PXE cohort, choroidal thickness was significantly reduced along the horizontal and vertical scans through the foveal center ( Figure 4 , left). Overall, there appeared to be a gradient of choroidal thinning in patients with PXE. Relative to normal controls, the choroid was thinnest close to the optic nerve head, approximating normal thickness values temporally along the horizontal OCT scan ( Figure 4 , top row). Along the vertical OCT scan there also was a gradient, with the most pronounced thinning at the foveal center and approximation to values of the control group with increasing eccentricity ( Figure 4 , bottom row). Comparison of the 3 PXE subgroups revealed the least choroidal thinning in Group 1 and the most pronounced thinning in Group 3.

Choroidal Structure in Pseudoxanthoma Elasticum

On cross-sectional EDI-OCT scans, choroidal structure in healthy middle-aged controls was characterized by a layer of small and medium-sized vessels in the inner choroid, accounting for about one third to one half of choroidal thickness, and larger vessels in the outer choroid ( Figure 5 , first row). Within all PXE subgroups, choroidal thinning appeared to affect the inner choroid primarily, with associated enhanced visibility of larger outer choroidal vessels. While choroidal thickness was largest subfoveally in normal controls ( Figure 5 , first row), eyes of patients with PXE revealed a gradient of marked thinning close to the optic disc to more preserved choroid temporally ( Figure 5 , second to fourth rows). Subretinal fluid accumulations were not accompanied by different changes of choroidal thickness or structure ( Figure 5 , last row).

Morphology of the choroid in patients with pseudoxanthoma elasticum (PXE). Enhanced-depth imaging optical coherence tomography (EDI-OCT) images (right; fundus position marked by the arrow in the left-hand corresponding reflectance image) show the choroidal thickness and cross-sectional structures in eyes of patients with PXE representative of the 3 different subgroups, as well as of 1 eye with PXE-associated subretinal fluid (SRF) in comparison to a representative age-matched healthy control (first row). The dashed line marks the sclerochoroidal junction in EDI-OCT images. Eyes of patients with PXE showed marked thinning of the choroid, which was most pronounced nasally (second-to-last row). Structural changes in the choroid included thinning or loss of the inner choroid, leading to enhanced and more anterior visibility of larger choroidal vessels (arrows in EDI-OCT scans). In Groups 2 and 3, choroidal vessels were virtually absent close to the optic disc (arrowheads). Note that choroidal changes in a patient with subretinal fluid accumulation (last row) were similar to those in patients in Group 1 (second row).

EDI-OCT volume-scans were used to reconstruct en face views 60 μm below the RPE where the inner choroid was well visualized in most healthy controls. In the first row of Figure 6 , a representative control eye illustrates the normal dense choroidal vascular network of the inner choroid with little spacing between vessels. In a Group 1 eye with PXE there was marked rarefication of this dense vascular network and increased visibility of larger choroidal vessels. Between vessels there was increased spacing with interposed hyperreflective material ( Figure 6 , second row). Patients in Group 2 exhibited a comparable choroidal structure and additional visibility of the CNV, which interfered with visualization of the choroid ( Figure 6 , third row). In the patients in Group 3, the choroid was almost completely absent close to the optic disc, whereas some larger vessels of the outer choroid were visible temporally ( Figure 6 , last row).

En-face enhanced-depth imaging optical coherence tomography (EDI-OCT) images of the choroid in pseudoxanthoma elasticum (PXE). Representative en-face EDI-OCT images (left) and corresponding EDI-OCT line scans (right) centered on the fovea in a healthy control and the 3 subgroups of PXE-related fundus changes. The dotted line in EDI-OCT line scans marks the position of the en face views 60 μm below Bruch membrane. The visibility of a dense vascular network in the controls (first row) was reduced in eyes of PXE patients, decreasing from Group 1 to Group 3 (second-to-last row) with visibility of larger choroidal vessels. The latter was sometimes interrupted by choroidal neovascularization and/or associated fibrosis (arrow in third row, left). In eyes with chorioretinal atrophy (Group 3) choroidal vessels were partly not visible anymore (last row). Scale bars = 200 μm.

Dependence on Age and Refractive Error

Choroidal thickness is negatively correlated with age in healthy subjects. It was of interest to discover whether age dependency of choroidal thickness was different in eyes with Bruch membrane impairment. Therefore, the subfoveal choroidal thickness of patients with PXE in Group 1 and of healthy controls was separately plotted against age ( Figure 7 , graph). Thickness measurements were negatively correlated with age within both groups (r = −0.42, P = 0.002; r = −0.612, P = 0.001). However, age-dependent choroidal thinning was significantly enhanced in patients with PXE compared to controls ( P < 0.001).

Age dependency of choroidal thickness in pseudoxanthoma elasticum (PXE). The graph represents subfoveal choroidal thickness plotted against age for controls and patients with PXE. Only Group 1 eyes with PXE were analyzed so as to avoid interactions due to choroidal neovascularization or chorioretinal atrophy. Age-related choroidal thinning was significantly enhanced in eyes of patients with PXE compared to controls ( p <0.001). Analysis of cross-sectional scans representative of 3 different age groups revealed accelerated choroidal thinning in eyes with PXE (right; 24, 39 and 55 years of age) compared to controls (left; 20, 39 and 56 years of age). Thinning typically began close to the optic disc, affecting more peripheral areas later in the disease course.

With regard to the topography of age-dependent choroidal thinning, controls showed a homogeneous thinning at different eccentricities ( Figure 7 , left) whereas in patients with PXE, thinning of the choroid started nasally and then spread to the temporal periphery with increasing age ( Figure 7 , right).

There was a tendency toward a positive correlation between refractive error and thickness that was comparable in all groups.