Ophthalmic Surgery, Lasers and Imaging Retina

Clinical Science 

Optical Coherence Tomography Angiography Findings in X-Linked Retinoschisis

Rodolfo Mastropasqua, MD; Lisa Toto, MD; Luca Di Antonio, MD; Maurizio Battaglia Parodi, MD; Luca Sorino, MD; Ivana Antonucci, MD; Liborio Stuppia, MD; Marta Di Nicola, MD; Cesare Mariotti, MD

Abstract

BACKGROUND AND OBJECTIVE:

The aim of the study was to determine optical coherence tomography angiography (OCTA) findings and to identify mutations in the RS1 gene in a three-generation family with X-linked juvenile retinoschisis (XLRS).

PATIENTS AND METHODS:

Clinical and genetic assessments were performed in 12 family members. OCTA was performed at baseline (12 members including cases and carriers) and after acetazolamide administration (three cases). Twenty healthy subjects (20 eyes, controls) were chosen for comparison. Molecular genetic analysis of the RS1 gene was performed in all family members.

RESULTS:

Deep capillary plexus density was reduced in cases compared with controls (P < .01) and was negatively related with retinal thickness (P < .05). After treatment, retinal thickness decreased (P < .05) and deep capillary plexus density increased (P < .05) in cases. In three cases and in four carriers, p.Arg197 His mutation was found.

CONCLUSION:

OCTA shows reduced macular deep vessel density in patients with XLRS probably related to vessel displacement and disruption due to schitic cysts.

[Ophthalmic Surg Lasers Imaging Retina. 2018;49:e20–e31.]

Abstract

BACKGROUND AND OBJECTIVE:

The aim of the study was to determine optical coherence tomography angiography (OCTA) findings and to identify mutations in the RS1 gene in a three-generation family with X-linked juvenile retinoschisis (XLRS).

PATIENTS AND METHODS:

Clinical and genetic assessments were performed in 12 family members. OCTA was performed at baseline (12 members including cases and carriers) and after acetazolamide administration (three cases). Twenty healthy subjects (20 eyes, controls) were chosen for comparison. Molecular genetic analysis of the RS1 gene was performed in all family members.

RESULTS:

Deep capillary plexus density was reduced in cases compared with controls (P < .01) and was negatively related with retinal thickness (P < .05). After treatment, retinal thickness decreased (P < .05) and deep capillary plexus density increased (P < .05) in cases. In three cases and in four carriers, p.Arg197 His mutation was found.

CONCLUSION:

OCTA shows reduced macular deep vessel density in patients with XLRS probably related to vessel displacement and disruption due to schitic cysts.

[Ophthalmic Surg Lasers Imaging Retina. 2018;49:e20–e31.]

Introduction

X-linked juvenile retinoschisis (XLRS) is one of the most common hereditary causes of juvenile vitreoretinal degeneration in males, associated with mutations in the retinoschisin gene (RS1, formerly XLRS1) located on the Xp22.1 chromosome, causing variable phenotype.1–5

Photoreceptors and bipolar cells are involved in the disease process. In fact, the RS1 gene encodes a 24-kDa discoidin domain-containing protein that is secreted as a homo-oligomeric complex. This complex binds tightly to the surface of photoreceptors and bipolar cells where it helps to maintain the cellular organization of the retina and structure of the photoreceptor-bipolar synapse.6, 7

XLRS is characterized by early onset visual loss and bilateral foveal schisis from the splitting of inner retinal layers, present in 98% to 100% of patients. In childhood, schitic cavities may be of bullous appearance; in older patients, cystic spaces can regress, leaving atrophic lesions. Peripheral retinoschisis can be present in the inferotemporal region in approximately 50% of patients.

Additional peripheral changes may include pigmentation, retinal fibrosis, vascular attenuation or sheathing, vitreous veils, and white retinal flecks.8

Retinoschisis develops in different layers, from the retinal nerve fiber layer (RNFL) to the outer nuclear layer (ONL), and the retinal vessels may lie in either the outer or inner leaf, sometimes crossing through the schisis cavity.9–11

Optical coherence tomography (OCT) is the most useful diagnostic tool in XLRS, providing detailed characterization of retinal morphological changes in the macular area.12

A new imaging modality, OCT angiography (OCTA), has recently been developed to study retinal and choroidal microvasculature without dye injection and has already been used to study the superficial and the deep retinal vascular plexuses and choriocapillaris (CC) in several retinal vascular diseases.13–15

We report genetic assessment and multimodal imaging of a three-generation family with XLRS, focusing on OCTA findings of the macula, and we compare vessel density and central macular thickness (CMT) in the macular area of family members with vessel density of normal controls.

Patients and Methods

Methods

Twelve members of a three-generation family with XLRS were examined at the Retina Service of the Ophthalmology Clinic, University “G. d'Annunzio,” Chieti, Italy. The study adhered to the tenets of the Declaration of Helsinki, and informed consent was obtained from all subjects. Approval of the institutional review board of University “G. d'Annunzio” of Chieti was received.

All family members underwent full clinical assessment and genetic analysis. Twenty healthy age-matched subjects (20 eyes) formed the control group.

Clinical evaluation in the family members included medical history, best-corrected visual acuity (BCVA) assessed using an Early Treatment Diabetic Retinopathy Study chart, funduscopy, color fundus photography, fundus autofluorescence (FAF), fundus fluorescein angiography (FFA) (Spectralis HRA+OCT; Heidelberg Engineering, Heidelberg, Germany), spectral-domain OCT (SD-OCT), and OCT angiography (OCTA) (XR Avanti AngioVue OCTA; Optovue, Fremont, CA).

Control cases underwent a complete ophthalmic evaluation including BCVA assessment, funduscopy, SD-OCT, and OCTA assessment.

In addition, all affected members underwent SD-OCT and OCTA after 2 months of administration of oral 250 mg acetazolamide tablets (Diamox; Teva Pharmaceuticals, Petah Tikva, Israel) twice daily and 2% dorzolamide collyrium (Trusopt; Santen Pharmaceutical, Osaka, Japan).

SD-OCT Angiography With XR Avanti

The XR Avanti AngioVue OCTA is a device with a high speed of 70,000 axial scans per second, using a light source of 840 nm and an axial resolution of 5 μm. The AngioVue OCTA system, based on split-spectrum amplitude-decorrelation angiography algorithm (software version 2017.1.0.129), uses blood flow as intrinsic contrast. Indeed, the flow is detected as a variation over time in the speckle pattern formed by interference of light scattered from red blood cells and adjacent tissue structures.13

Before imaging, the pupils of each subject were dilated with a combination of 0.5% tropicamide and 10% phenylephrine. Study participants underwent SD-OCT imaging following a protocol that included AngioVue OCT 3-D volume set of 3 mm × 3 mm. An internal fixation light was used to center the scanning area.

One FastX (horizontal raster) set and one FastY (vertical raster) set were performed for each acquisition scan. Scans with low quality (ie, if the subject blinked or if there were significant motion artifacts) were excluded and repeated until good quality was achieved. Three scans for each patient were captured (all with a signal straight index > 60), and the scan of best quality was chosen for analysis.

Vascular Layer Segmentation

Vascular retinal layers were visualized and segmented based on the default settings of the automated software algorithm of the XR Avanti AngioVue OCTA. The superficial plexus consists of the capillaries 3 μm below the internal limiting membrane (ILM) to 15 μm below the inner plexiform layer (IPL). The deep plexus extends from 15 μm to 70 μm below the IPL. The CC consists of capillaries in a 30 μm thick layer posterior to the retinal pigment epithelium-Bruch membrane junction.

The three-dimensional projection artifacts removal (3-D PAR) is a new algorithm able to simplify interpretation of OCTA images, enhancing depth resolution of vascular layers. This modality allows the retention of flow signal from real blood vessels while suppressing projected flow signal in deeper layers, avoiding downward tails on cross-sectional angiograms and duplicated vascular patterns on en face angiograms. As previously described, the algorithm identifies voxel with “in situ” flow as those in which intensity-normalized decorrelation values are higher than all shallower voxel in the same axial position.16

In addition, two investigators (LT and LDA) checked the segmentation quality and were able to finely adjust the slab positioning in order to ensure a correct visualization of both the superficial and the deep vascular plexuses. The images were reviewed by two investigators for correctness of segmentation; if segmentation errors were observed, they were corrected using segmentation editing and propagation tool embedded in the AngioVue system. The final image was reviewed again to confirm the segmentation placement through all the B-scans and was chosen only if a consensus was achieved between the two investigators.

Vessel Density Analysis

Objective quantification vessel density (VD) was evaluated on the OCTA en face images for each eye using the AngioVue software. The flow area was calculated with a user-defined circular region of interest (ROI) and a threshold. The area within the ROI with intensities greater than the threshold was calculated. The VD was defined as the percentage area occupied by vessels in a circular ROI centered on the center of the foveal avascular zone and with a diameter of 2.5 mm (whole area). The AngioVue software automatically splits the ROI into two fields: the foveal area, a central circle with a diameter of 1 mm, and the parafoveal area that constitutes the remaining part inside the ROI.

The AngioVue software automatically outputs the vessel density percentage inside the foveal area, in the whole parafoveal area, in the hemi superior and inferior fields, and in different quadrants of the parafoveal area. Foveal and parafoveal density of superficial and deep capillary plexuses and of CC were analyzed.

The vessel density is calculated using the formula previously described,13 as follows: Vessel density = ∫ V∙dA/∫ dA, in which V is 1 when the OCTA value is above a background threshold and 0 otherwise. A is the area of interest (Figure 1).

Optical coherence tomography (OCT) angiography scan of 3 mm × 3 mm with superimposed circular region of interest centered on the center of the foveal avascular zone and with a diameter of 2.5 mm of the superficial plexus, deep plexus, and choriocapillaris (A, B, C, top panel) and corresponding spectral OCT (A, B, C, middle panel) and perfusion maps (A, B, C, bottom panel).

Figure 1.

Optical coherence tomography (OCT) angiography scan of 3 mm × 3 mm with superimposed circular region of interest centered on the center of the foveal avascular zone and with a diameter of 2.5 mm of the superficial plexus, deep plexus, and choriocapillaris (A, B, C, top panel) and corresponding spectral OCT (A, B, C, middle panel) and perfusion maps (A, B, C, bottom panel).

Central Retinal Thickness Analysis

Foveal retinal thickness (RT), parafoveal RT, parafoveal RT in the superior and inferior hemi macular area (S-Hemi RT and I-Hemi RT) from the ILM to the retinal pigment epithelium (ILM-RPE) (whole retina), and parafoveal RT and S-Hemi and I-Hemi RT from ILM to IPL (inner retina) were automatically calculated by the software on the OCTA 3 mm × 3 mm volume scan (XR Avanti). A circular ROI centered on the center of the foveal avascular zone with a diameter of 3.0 mm was considered for retinal thickness analysis: central foveal area (1 mm of diameter) and parafoveal area that constitutes the remaining part inside the ROI (full parafoveal area or temporal, superior, nasal, and inferior quadrants).

Genetic Analysis

Genetic investigation consisted of genomic DNA extraction (12 family members) from peripheral blood using a BioRobot EZ1 instrument (Qiagen, Milan, Italy), according to the manufacturer's protocol, at the Molecular Genetics service of Chieti University. Amplification of all coding exons and of each flanking intron of gene was performed using polymerase chain reaction (PCR) followed by direct DNA sequencing. The entire coding region comprised of six exons, and the flanking intronic regions of the RS1 gene were amplified with PCR using specific primers (available on request).

Statistical Analysis

The quantitative variables were summarized as median and interquartile range, according to their distribution. Qualitative variables were summarized as frequency and percentage. Shapiro-Wilk's test was performed to evaluate the departures from normality distribution for each variable. Kruskal-Wallis H-test was performed to evaluate differences in functional and morphological parameters between cases, controls, and carriers groups. Spearman's rho correlation coefficient was evaluated to assess correlation among thickness parameters and density parameters in the cases group. Wilcoxon signed rank test was performed to evaluate the difference between baseline parameters and post-treatment parameters in the cases group. The false discovery rate (FDR) correction was performed to control the family-wise type I error rate, and an FDR adjusted P value less than .05 was determined to be statistically significant. Statistical analysis was performed using IBM SPSS Statistics version 20.0 software (SPSS, Chicago, IL).

Results

Clinical Findings of the Family Members

Twelve family members from one family of European ethnicity were examined. The pedigree is shown in Figure 2.

Pedigree of the family.

Figure 2.

Pedigree of the family.

Patients (three members) were diagnosed with XLRS based on clinical and genetic assessment. In all affected members, ophthalmoscopy showed bilateral macular schisis with spoke-like pattern. SD-OCT confirmed the presence of foveal schisis with cystoid spaces involving different layers from the RNFL to the ONL. FAF revealed an irregularly shaped area of hyper- and hypo-FAF or spoke-wheel pattern of hyper- and hypo-FAF. FFA showed that macular schisis cavities were not associated with late leakage. OCTA showed interruption of integrity of the perifoveal anastomotic arcades in superficial capillary plexus (SCP) and vessel rarefaction in the deep capillary plexus (DCP) and presence of cystoid spaces mainly in the DCP.

The proband (patient III:3), a 16-year-old Caucasian male, was first seen by an ophthalmologist at the age of 8 years, and a clinical diagnosis of XLRS was made. When we examined him at age 16, his BCVA was 0.4 logMAR (20/50) in both eyes. On biomicroscopy, the anterior segment was unremarkable. Biomicroscopic examination of the fundus showed a spoke-wheel pattern of fold-like changes radiating out from the fovea (Figure 3A, top panel). FAF showed a spoke-wheel pattern of hyper- and hypo-FAF in both eyes (Figure 3A, bottom panel). FFA showed that macular schisis cavities were not associated with late leakage (Figure 3B). OCTA showed interruption of integrity of the perifoveal anastomotic arcades in SCP and macular vessel rarefaction in DCP due to schitic cysts. Presence of no flow areas of cystoid appearance was present in the SCP and mainly in the DCP in a radiating pattern in the foveal and parafoveal areas (Figures 3C and 3D, top and middle panels). SD-OCT images from the vertical and horizontal scans centered on the fovea showed schisis in the ganglion cell layer (GCL) in the parafoveal region and in the inner nuclear layer (INL) from the foveal to the extrafoveal region. Schitic cystoid spaces were few and of small dimensions in the GCL and large partially coalescent in the INL (Figures 3C and 3D, bottom panel). The patient agreed to administration of oral 250 mg acetazolamide tablets twice daily and 2% dorzolamide collyrium for 2 months. After the 2 months of therapy, central retinal thickness (CRT) changed from 355 μm to 178 μm in the right eye and from 400 μm to 265 μm in the left eye, and visual acuity (VA) improved to 0.3 logMAR in the right eye and 0.4 logMAR in the left eye. On OCTA in both eyes in the DCP, there was a partial increase of vessel density and decrease of cystoid spaces (Figure 4).

Images of the left eye of the proband (III:3). Color fundus photograph showing a spoke-wheel pattern of fold-like changes radiating out from the fovea (A, top panel). Fundus autofluorescence (FAF) showing spoke-wheel pattern of hyper- and hypo-FAF (A, bottom panel). Early and late fundus fluorescein angiography (FFA) showing macular schisis cavities were not associated with late leakage (B, top and bottom panels). Optical coherence tomography (OCT) angiography of the superficial plexus (B, top panel) and deep plexus (C, top panel) with corresponding perfusion vessel density maps (B and C, middle panels, respectively) showing vessel rarefaction in the superficial plexus and deep plexus with no flow areas of cystoid appearance in the deep plexus. Corresponding structural OCT horizontal scan spectral-domain OCT images from the vertical and horizontal scans centered on the fovea showed schisis in the ganglion cell layer in the parafoveal region and in the inner nuclear layer from the foveal to the extrafoveal region (B and C, bottom panel).

Figure 3.

Images of the left eye of the proband (III:3). Color fundus photograph showing a spoke-wheel pattern of fold-like changes radiating out from the fovea (A, top panel). Fundus autofluorescence (FAF) showing spoke-wheel pattern of hyper- and hypo-FAF (A, bottom panel). Early and late fundus fluorescein angiography (FFA) showing macular schisis cavities were not associated with late leakage (B, top and bottom panels). Optical coherence tomography (OCT) angiography of the superficial plexus (B, top panel) and deep plexus (C, top panel) with corresponding perfusion vessel density maps (B and C, middle panels, respectively) showing vessel rarefaction in the superficial plexus and deep plexus with no flow areas of cystoid appearance in the deep plexus. Corresponding structural OCT horizontal scan spectral-domain OCT images from the vertical and horizontal scans centered on the fovea showed schisis in the ganglion cell layer in the parafoveal region and in the inner nuclear layer from the foveal to the extrafoveal region (B and C, bottom panel).

Images of the right eye of the proband (III:3) before and after treatment with oral 250 mg acetazolamide tablets twice daily and 2% dorzolamide collyrium. Spectral-domain optical coherence tomography (OCT) images from the vertical and horizontal scans centered on the fovea showing schisis in the ganglion cell layer in the parafoveal region and in the inner nuclear layer from the foveal to the extrafoveal region (A, top panel). En face OCT showing schitic cysts in a radial pattern in the foveal and parafoveal areas before treatment (B, top panel). Corresponding OCT angiography (OCTA) perfusion maps showing vessel density reduction in the foveal and parafoveal areas in the superficial and deep capillary plexuses (C and D, top panel, respectively). After treatment, there is a reduction of central retinal thickness (A, bottom panel) on spectral-domain OCT and partial resolution of schitic cysts particularly in the temporal area (B, bottom panel) on en face OCT. Vessel density partially increases after treatment on OCTA (C and D, top and bottom panels).

Figure 4.

Images of the right eye of the proband (III:3) before and after treatment with oral 250 mg acetazolamide tablets twice daily and 2% dorzolamide collyrium. Spectral-domain optical coherence tomography (OCT) images from the vertical and horizontal scans centered on the fovea showing schisis in the ganglion cell layer in the parafoveal region and in the inner nuclear layer from the foveal to the extrafoveal region (A, top panel). En face OCT showing schitic cysts in a radial pattern in the foveal and parafoveal areas before treatment (B, top panel). Corresponding OCT angiography (OCTA) perfusion maps showing vessel density reduction in the foveal and parafoveal areas in the superficial and deep capillary plexuses (C and D, top panel, respectively). After treatment, there is a reduction of central retinal thickness (A, bottom panel) on spectral-domain OCT and partial resolution of schitic cysts particularly in the temporal area (B, bottom panel) on en face OCT. Vessel density partially increases after treatment on OCTA (C and D, top and bottom panels).

Patient II:1, uncle of the proband, a 44-year-old white male, was first diagnosed as having XLRS by an ophthalmologist at the age of 30 years. His BCVA was 0.4 logMAR (20/50) in both eyes. On biomicroscopy, the anterior segment was normal. Biomicroscopic fundus examination showed a spoke-wheel pattern of fold-like changes radiating out from the fovea. FAF showed an irregularly shaped area of hyper- and hypo-FAF in the right eye and a spoke-wheel pattern of hyper- and hypo-FAF in the left eye. OCTA showed interruption of integrity of the perifoveal anastomotic arcades in SCP and macular vessel rarefaction in DCP. Presence of no flow areas of cystoid appearance were present in the SCP and mainly in the DCP in a radiating pattern in the foveal and parafoveal areas. SD-OCT images from the vertical and horizontal scans centered on the fovea showed schisis in the GCL in the parafoveal region, in the INL from the foveal to the extrafoveal region, and in the outer plexiform layer (OPL) from the foveal to the extrafoveal region. Schitic cystoid spaces were few and of small dimension in the GCL. In the INL and OPL, the schitic cystoid spaces were large and partially coalescent in the foveal region and numerous with decreasing dimension from the parafoveal to the extrafoveal region (Figures 4B and 4C, bottom panels). The patient agreed to administration of oral 250 mg acetazolamide tablets twice daily and 2% dorzolamide collyrium for 2 months. After the 2 months of therapy, CRT decreased from 512 μm to 288 μm in the right eye and from 492 μm to 285 μm in the left eye, and VA improved to 0.3 logMAR in both eyes. On OCTA in the DCP, there was a vessel density redistribution and decrease of cystoid spaces.

Patient II:2, uncle of the proband, a 47-year-old white male, was diagnosed by an ophthalmologist with “macular dystrophy” at the age of 16 years, and a diagnosis of XLRS was made at age of 36 years. When we examined him, his BCVA was 0.7 logMAR in both eyes. On biomicroscopy, the anterior segment was normal. Biomicroscopic examination of the fundus showed a spoke-wheel pattern of fold-like changes radiating out from the fovea and some yellow flecks at the posterior pole interspersed with some dots of calcific appearance mainly localized in the macula region. Pallor of the optic disc was present in both eyes associated with partial vascular sheathing in the peripapillary area of the left eye. FAF showed a spoke-wheel pattern of hyper- and hypo-FAF in both eyes and high hyper-FAF small lesions related to flecks and calcified lesions. OCTA showed enlargement of the foveal avascular zone in SCP and macular vessel rarefaction in DCP. Presence of no-flow areas of cystoid appearance was present in the SCP and mainly in the DCP in a radiating pattern in the foveal and parafoveal areas. SD-OCT images from the vertical and horizontal scans centered on the fovea showed schisis in the GCL in the parafoveal region, in the INL from the foveal to the extrafoveal region, and in the OPL from the foveal to the extrafoveal region. Schitic cystoid spaces were few and of small dimension in the GCL, but in the INL and OPL were large and coalescent in the foveal region and numerous with decreasing dimension from the parafoveal to the extrafoveal region. Schisis showed greater extension in the inferior extrafoveal quadrant approaching the inferior vascular arcade. The patient agreed to administration of administration of oral 250 mg acetazolamide tablets twice daily and 2% dorzolamide collyrium for 2 months. After the 2 months of therapy, CRT decreased from 550 μm to 348 μm to in the right eye and from 348 μm to 287 μm in the LE, and VA improved to 0.6 logMAR in both eyes. On OCTA in the DCP, there was a vessel density redistribution and decrease of cystoid spaces.

Family carrier members I:2, II:4, III:1, and III:2 did not show any retinal alterations.

Genetic Findings of the Family Members

DNA sequencing of the RS1 gene in three affected male patients and their available family members identified a missense mutation in the exon 6, leading to an arginine to histidine change at amino acid position 197 (p.Arg197 His) (Figure 5).

Sequencing electropherograms showing p.Arg197 His mutation in exon 6 of RS1 gene.

Figure 5.

Sequencing electropherograms showing p.Arg197 His mutation in exon 6 of RS1 gene.

Three male patients of this family were identified as having the Arg197 His mutation whereas four female carriers showed heterozygous patterns, including both wild-type and mutant alleles.

No additional mutations were detected in other coding regions.

Demographic Data of Family Members Compared With Controls

The mean age was 35.7 years (range: 16 years to 47 years) for cases (affected family members), 35.5 years (range: 13 years to 68 years) for carriers, and 36.7 years (range: 15 years to 47 years) years for healthy controls (P = .983 for cases vs. carriers and P = .884 for cases vs. healthy controls).

Central Retinal Thickness Analysis of Family Members Compared With Controls

ILM-RPE foveal RT, ILM-RPE parafoveal RT, and ILM-RPE I-Hemi RT were significantly increased in cases compared with carriers and controls (P < .05) (Table 1). ILM-IPL parafoveal RT and ILM-IPL S-Hemi RT and I-Hemi RT were significantly increased in cases compared with carriers and controls (P < .05) (Table 1).

Morphological Parameters, Expressed as Median and Interquartile Range, in Cases, Carriers, and Controls

Table 1:

Morphological Parameters, Expressed as Median and Interquartile Range, in Cases, Carriers, and Controls

Vessel Density Analysis of Family Members Compared With Controls

Whole vessel density of the superficial plexus was significantly different in cases compared with controls (Table 1). In the deep plexus, whole vessel density and vessel density of different sectors were significantly reduced in cases compared with controls (Table 1). No significant differences of CC density were evidenced between cases and controls. No significant differences were found between carriers and controls in the superficial plexus, deep plexus and CC.

Correlation Analysis in Affected Members

Table 2 shows the results of the correlation analysis. Deep plexus density was significantly negatively related with ILM-IPL parafoveal RT, and parafoveal CC density was significantly negatively related with ILM-RPE parafoveal RT (Table 2).

Correlation Analysis Among Retinal Thickness Parameters and Vessel Density Parameters Assessed by Spearman Rho Correlation Coefficient in Affected Patients

Table 2:

Correlation Analysis Among Retinal Thickness Parameters and Vessel Density Parameters Assessed by Spearman Rho Correlation Coefficient in Affected Patients

Post-Treatment Quantitative Analysis

The three affected patients (six eyes) underwent administration of oral 250 mg acetazolamide tablets twice daily and 2% dorzolamide collyrium for 2 months. After treatment, ILM-RPE foveal and parafoveal RT and ILM-IPL parafoveal RT showed a significant decrease (P < .05), and vessel density showed a significant increase in the parafoveal area (P < .05) in the DCP (Table 3). The whole CC density showed a significant increase after treatment (P < .05).

Baseline and Post-Treatment Parameters, Expressed as Median and Interquartile Range

Table 3:

Baseline and Post-Treatment Parameters, Expressed as Median and Interquartile Range

Discussion

A range of vascular alterations has been described in the peripheral retina of XLRS patients characterized by vascular sheathing, vascularized veils in the vitreous, vitreous hemorrhage, dendritic vasculature in the peripheral retina, retinal neovascularization, peripheral avascular zones, and peripapillary choroidal neovascularization.17–19

Histological studies also demonstrated the presence of sclerotic vessels in both the inner and outer layers of the retina in the macula.20

The presence of vascular alterations and particularly of retinal ischemia and of peripheral avascularity of non-schitic areas mainly supported by the fluorescein angiographic findings and the histologic evidence of sclerotic macular vessels lead to the hypothesis of a diffuse retinal microangiopathy in XLRS patients that can also stimulate neovascularization.18

The use of OCTA in several retinal diseases allowed the study of retinal vasculature without dye injection and with detailed morphology.15, 21–25

In this study, the affected members of the three-generation family with XLRS and p.Arg197 His mutation were studied with a multimodal imaging approach focusing on OCTA findings in the macular area.

A variable clinical phenotype was observed in the affected members. On SD-OCT, schitic cystoid spaces were present in the GCL, INL, and OPL in two members (II:1 and II:2) and only in the GCL and INL in the third member (III:3). Cystic spaces were few and small in the GCL; in the INL and OPL they were large and partially coalescent in the foveal region and numerous with dimension reducing from the parafoveal to the extrafoveal region.

In one member (II:2), small flecks were visible at the posterior pole with some calcifications in the macular region appearing highly hyperfluorescent on FAF. The same patient at biomicroscopy showed vascular sheathing of the peripapillary inferotemporal vessels.

OCTA disclosed extensive vascular alterations in the macular area. In all affected members, an interruption of the perifoveal anastomotic arcades was particularly evident in the SCP, and vessel rarefaction was noticed in the deep plexus in the foveal and parafoveal areas. No flow areas of cystoid appearance were present in the foveal and parafoveal regions in the superficial plexus and many in the deep plexus with a spoke-like pattern related to schitic cysts. When a comparison was made between affected members, carriers, and healthy subjects, a significant reduction of whole vessel density of SCP and whole vessel density and vessel density of different sectors in the DCP was found in cases compared with controls.

ILM-RPE retinal thickness and ILM-IPL retinal thickness were significantly increased in cases compared with carriers and controls (P < .05).

A negative significant correlation was found in cases between vessel density and CRT.

After treatment, ILM-RPE retinal thickness and ILM-IPL retinal thickness showed a significant decrease (P < .05), and vessel density showed a significant increase mainly in the parafoveal area (P < .05) in the DCP.

The reduced vascular density in the foveal and parafoveal areas observed in patients with XLRS in the presence of schitic cysts could be related to vessel displacement. In addition, as already suggested for vascular alterations in myopic retinoschisis, the superficial and deep retinal capillary networks could be physically disrupted as the schisis cavity enlarges.26 The no complete vessel repopulation even after post-treatment disappearance of schitic cysts could be related to vessel damage due to schitic lesions. Nevertheless, as previously suggested by other authors, a primitive microangiopathy cannot be excluded.17–20

Retinoschisin (RS1) is a highly conserved 23 kDa extracellular protein consisting of four distinct regions: a 23 amino acid N-terminal signal sequence, a dominant 157 amino acid discoidin domain, a 39 amino acid Rs1 domain upstream of the discoidin domain, and a 5 amino acid C-terminal segment.27

Discoidin domain-containing proteins are widely distributed in eukaryotes and mediate a variety of functions, including cell adhesion, cell–extracellular matrix interactions, signal transduction, phagocytosis of apoptotic cells, axon guidance, angiogenesis, and blood clotting. Many of these proteins are involved in extracellular matrix or cell binding, although some bind ligands such as vascular endothelial growth factor and semaphorin.28 The function of retinoschisin in the retina is not completely known. It has been suggested that retinoschisin functions as a cell adhesion protein to maintain the cellular organization of the retina and the structural integrity of the photoreceptor-bipolar synapse. Another possible role of retinoschisin may be to help regulate the fluid balance between the intracellular and extracellular environment particularly within the photoreceptor and bipolar cell layers; thus, a loss of functional retinoschisin could cause fluid accumulation in the extracellular environment in the form of a fluid-filled cystic cavity.27 The cystic cavities could in turn disrupt the organized layers of the retina, causing a dysfunction of the photoreceptor-bipolar synapse.29

These hypotheses do not completely explain the role of retinoschisin in vascular alterations associated with XLRS. The diffuse microangiopathy could be secondary to retinal integrity disorganization, but this theory could not explain the presence of vascular anomalies also in no-schitic areas. A direct correlation with vascular anomalies should be considered; thus, a role of retinoschisin in vascular integrity should be explored.

X-linked retinoschisis is associated with several mutations with variable intrafamilial and interfamilial phenotypic expression. Some of them are nonsense or frameshift mutations that are predicted to result in the absence of a full-length retinoschisin protein or missense mutations that allow the translation of the full-length mutant protein.30,31

In our family, the three affected members showed a missense mutation (p.Arg197 His) with somewhat different phenotypes between the affected members as already demonstrated in other studies.30,31

A response to carbonic anhydrase inhibitors (CAIs) has been reported in patients affected by XLRS with partial or complete disappearance of cystoid macular edema under prolonged oral and/or topical therapy.32,33

The members of our family undergoing CAI treatment showed marked resolution of macular cysts with partial recovery of macular vessel disposition.

One of the main limits of the study is the limited number of patients included and analyzed because of carrying out the study on a single three-generation family with only three affected members. Probably a multicenter study on a larger case series at baseline and eventually after treatment also with different XLRS mutations would disclose variable phenotypes and possibly a different pattern of vessel alterations at OCTA. In addition, the analysis of larger retinal areas using OCTA scans with increased field of view could evidence possible vascular alterations outside the schitic cysts, confirming a more extensive retinal microangiopathy.

In conclusion, OCTA showed in detail vascular alterations associated with schitic cysts in all three XLRS-affected members, characterized by interruption of perifoveal anastomotic arcade in the superficial plexus and vessel rarefaction in the deep plexus. After oral and topic CAI treatment, partial vessel repopulation occurred.

References

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Morphological Parameters, Expressed as Median and Interquartile Range, in Cases, Carriers, and Controls

CasesCarriersControlsKruskal-WallisaP Value

Retinal Thickness (ILM-RPE)
  Fovea448.0 (407.5–552.8)254.0 (249.0–270.5)***249.0 (238.0–266.0)°°°.001
  Parafovea380.0 (332.0–389.5)309.5 (305.5–324.8)*314.0 (299.0–324.0)°.017
  S-Hemi367.5 (292.3–399.8)315.0 (309.0–321.0)314.0 (299.0–324.0).298
  I-Hemi352.5 (334.5–388.0)310.5 (303.0–326.5)*310.0 (300.0–328.0)°°.003

Retinal Thickness (ILM-IPL)
  Parafovea147.0 (134.8–152.3)121.0 (109.5–133.3)*118.0 (112.0–123.0)°°.002
  S-Hemi147.0 (136.5–153.0)119.5 (107.5–132.0)*118.0 (110.0–122.0)°°.004
  I-Hemi147.5 (133.3–149.3)121.5 (113.0–134.3)*118.0 (113.0–124.0)°°.002

Density of Superficial Plexus
  Whole44.1 (43.1–45.8)45.8 (44.1–45.7)45.7 (45.8–51.1)°°.004
  Fovea27.2 (23.0–42.4)42.4 (27.2–29.5)29.5 (42.4–33).730
  Parafovea47.5 (45.9–48.7)48.7 (47.5–46.1)46.1 (48.7–52.4).061
  Para-Superior Hemi47.6 (46.2–48.5)48.5 (47.6–45.4)45.4 (48.5–52.8).065
  Para-Inferior Hemi48.0 (46.6–48.8)48.8 (48–46.9)46.9 (48.8–51.9).081

Density of Deep Plexus
  Whole37.7 (36.7–39.8)57.2 (53.5–58.3)53.5 (39.8–57.2)°°°< .001
  Fovea32.2 (30.3–33.9)31.9 (30.6–35.6)30.6 (33.9–31.9).198
  Parafovea38.3 (37.7–38.6)59.9 (54.8–61.1)54.8 (38.6–59.9)°°°< .001
  Para-Superior Hemi38.4 (37.4–39.2)60.1 (55.0–62.0)55 (39.2–60.1)°°°.001
  Para-Inferior Hemi37.9 (36.7–38.9)58.9 (54.6–61.0)54.6 (38.9–58.9)°°°< .001

Density of Choriocapillary
  Whole64.6 (63.6–67.1)67.1 (64.6–64.9)64.9 (67.1–66.5).127
  Fovea69.2 (66.6–72.2)72.2 (69.2–65.6)65.6 (72.2–67.4).233
  Parafovea64.5 (59.3–66.1)66.1 (64.5–63.4)63.4 (66.1–66).289
  Para-Superior Hemi64.3 (59.8–66.3)66.3 (64.3–65)65 (66.3–65.4).497
  Para-Inferior Hemi64.7 (58.9–65.7)65.7 (64.7–63.2)63.2 (65.7–66).127

Correlation Analysis Among Retinal Thickness Parameters and Vessel Density Parameters Assessed by Spearman Rho Correlation Coefficient in Affected Patients

ILM-RPEILM-IPL


FoveaParafoveaS-HemiI-HemiParafoveaS-HemiI-Hemi

Density of Superficial Plexus
  Whole−0.714−0.771−0.257−0.714−0.486−0.257−0.551
  Fovea0.0860.371−0.0860.0860.0860.0290.010
  Parafovea−0.029−0.086−0.600−0.029−0.714−0.600−0.667
  Para-Superior Hemi0.2570.143−0.4860.257−0.543−0.429−0.464
  Para-Inferior Hemi−0.143−0.086−0.886−0.143−0.943*−0.886*−0.899

Density of Deep Plexus
  Whole−0.714−0.600−0.771−0.714−0.886*−0.771−0.928*
  Fovea−0.714−0.771−0.314−0.714−0.486−0.314−0.522
  Parafovea−0.058−0.29−0.232−0.058−0.377−0.261−0.294
  Para-Superior Hemi−0.143−0.314−0.086−0.143−0.314−0.486−0.203
  Para-Inferior Hemi−0.058−0.058−0.058−0.0580.0290.319−0.044

Density of Choriocapillary
  Whole−0.829−0.8860.143−0.829−0.086−0.143−0.116
  Fovea−0.143−0.257−0.486−0.143−0.543−0.486−0.464
  Parafovea−0.886−0.943*0.086−0.886−0.200−0.257−0.232
  Para-Superior Hemi−0.886−0.943*0.086−0.886−0.200−0.257−0.232
  Para-Inferior Hemi−0.886−0.943*0.086−0.886−0.200−0.257−0.232

Baseline and Post-Treatment Parameters, Expressed as Median and Interquartile Range

BaselinePost-TreatmentWilcoxon Signed Rank P Value

Retinal Thickness (ILM-RPE)
  Fovea448.0 (407.5–552.8)312.5 (262.0–350.3).028
  Parafovea380.0 (332.0–389.5)300.5 (262.0–331.5).026
  S-Hemi367.5 (292.3–399.8)311.0 (277.5–338.5).093
  I-Hemi352.5 (334.5–388.0)296.0 (266.5–322.5).027

Retinal Thickness (ILM-IPL)
  Parafovea147.0 (134.8–152.3)116.0 (101.8–125.0).028
  S-Hemi147.0 (136.5–153.0)114.0 (103.0–124.3).025
  I-Hemi147.5 (133.3–149.3)118.5 (101.3–126.0).030

Density of Superficial Plexus
  Whole44.1 (43.1–45.8)45.9 (45.1–46.3).273
  Fovea27.2 (23.0–42.4)26.7 (24.6–32.3).751
  Parafovea47.5 (45.9–48.7)48.0 (47.3–49.3).654
  Para-Superior Hemi47.6 (46.2–48.5)47.9 (46.3–49.7).871
  Para-Inferior Hemi48.0 (46.6–48.8)48.7 (48.4–48.9).756

Density of Deep Plexus
  Whole37.7 (36.7–39.8)49.9 (46.5–54.1).030
  Fovea32.2 (30.3–33.9)32.4 (31.1–35.8).456
  Parafovea38.3 (37.7–38.6)49.4 (46.3–54.3).050
  Para-Superior Hemi38.4 (37.4–39.2)47.5 (45.4–55.8).050
  Para-Inferior Hemi37.9 (36.7–38.9)51.5 (46.2–53.7).050

Density of Choriocapillary
  Whole64.6 (63.6–67.1)67.1 (66.2–67.8).050
  Fovea69.2 (66.6–72.2)67.9 (64.6–69.6).144
  Parafovea64.5 (59.3–66.1)66.4 (66.0–67.3).144
  Para-Superior Hemi64.3 (59.8–66.3)66.0 (65.7–67.6).465
  Para-Inferior Hemi64.7 (58.9–65.7)66.8 (66.1–67.1).050
Authors

From Ophthalmology Clinic, University of Marche, Ancona, Italy (RM, CM); Ophthalmology Clinic, Department of Medicine and Science of Ageing, University “G. d'Annunzio” Chieti-Pescara, Chieti, Italy (LT, LDA); the Department of Ophthalmology, University Vita-Salute, Scientific Institute San Raffaele, Milano, Italy (MBP); the Department of Oral Sciences, Nano and Biotechnologies, University “G. d'Annunzio” Chieti- Pescara, Chieti, Italy (LS, IA, L Stuppia); and the Laboratory of Biostatistics, Department of Medical, Oral and Biotechnological Sciences, University “G. d'Annunzio” Chieti- Pescara, Chieti, Italy (MDN).

The authors report no relevant financial disclosures.

Drs. Mastropasqua and Toto contributed equally to the manuscript as co-first authors.

Address correspondence to Lisa Toto, MD, Via dei Vestini 66100 Chieti, Italy; email: l.toto@unich.it.

Received: October 19, 2017
Accepted: January 22, 2018

10.3928/23258160-20180907-03

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