Multimodal Retinal Imaging and Microperimetry Reveal a Novel Phenotype and Potential Trial End Points in CRB1-Associated Retinopathies

Abstract

Purpose: Biallelic crumbs cell polarity complex component 1 (CRB1) mutations can present as Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), or cystic maculopathy. This study reports a novel phenotype of asymptomatic fenestrated slit maculopathy (AFSM) and examines macular volume profile and microperimetry as clinical trial end points in CRB1-associated retinopathies.

Methods: Twelve patients from nine families with CRB1 mutation were recruited. Ultra-widefield (UWF) color fundus photography and autofluorescence (AF), spectral-domain optical coherence tomography (SD-OCT), microperimetry, and adaptive optics (AO) imaging were performed. Macular volume profiles were compared with age-matched healthy controls. Genotyping was performed using APEX genotyping microarrays, targeted next-generation sequencing, and Sanger sequencing.

Results: We identified one patient with LCA, five patients with RP, and four patients with macular dystrophy (MD) with biallelic CRB1 mutations. Two siblings with compound heterozygote genotype (c.[2843G>A]; [498_506del]) had AFSM characterized by localized outer retinal disruption on SD-OCT and parafoveal cone loss on AO imaging despite normal fundus appearance, visual acuity, and foveal sensitivity. UWF AF demonstrated preserved para-arteriolar retinal pigment epithelium (PPRPE) in all patients with RP. Microperimetry documented preserved central retinal function in six patients. The ratio of perifoveal-to-foveal retinal volume was greater than controls in 89% (8/9) of patients with RP or MD, whereas central subfield and total macular volume were outside normal limits in 67% (6/9).

Conclusions: AO imaging was helpful in detecting parafoveal cone loss in asymptomatic patients. Macular volume profile and microperimetry parameters may have utility as CRB1 trials end points.

Translational Relevance: Macular volume and sensitivity can be used as structural and functional end points for trials on CRB1-associated RP and MD.

Introduction

Crumbs cell polarity complex component 1 (CRB1; OMIM: #604210) is a transmembrane protein, located within the subapical region of human photoreceptors that plays a critical role in retinal development, structure, and function.^1,2 Biallelic mutations in CRB1 have been implicated in 3% to 9% of autosomal recessive retinitis pigmentosa (arRP), including retinitis pigmentosa 12 (RP12; OMIM: #600105)³ and 7% to 17% of Leber congenital amaurosis (LCA), also known as LCA8 (OMIM: #613835).^4,5 Maculopathy with or without foveal schisis is a more recently recognized phenotype associated with CRB1 mutations.^6–11 Given gene therapy for CRB1-associated retinopathy is already underway in an animal model,¹² it is timely to examine the various imaging biomarkers that could be considered in future CRB1 treatment trials. 

CRB1-associated retinitis pigmentosa (CRB1-RP) has been associated with coarse retinal lamination, thick retina, preserved para-arteriolar retinal pigment epithelium (PPRPE), hypermetropia, cystoid macular edema (CMO), and retinal telangiectasia with Coat's-like reaction.¹³ For example, the likelihood of detecting CRB1 mutation in patients with RP is higher among those presenting with PPRPE (66%–74%) or retinal telangiectasia (31%–53%).⁴ Jacobson and colleagues reported abnormal parafoveal retinal thickening in eight patients with CRB1-associated LCA or early-onset severe RP.¹⁴ Their study used low resolution time-domain optical coherence tomography (OCT) cross section images without thickness mapping and only a relatively small number of healthy control subjects were recruited (n = 3). In a cohort of 55 patients with CRB1-RP, Talib et al. reported perifoveal retinal thickening in 82% (9/11) of patients and PPRPE in 26% (13/50) and 33% (3/9) of patients on widefield color composite fundus photographs and fundus autofluorescence (FAF) imaging, respectively.¹⁵ Although these cases were followed for 15.4 years, adaptive optics (AO) imaging and microperimetry data were not presented. Whereas universal functional markers, such as full-field stimulus and pupillography, may be used as trial end point in patients with late-stage RP caused by CRB1 mutations,¹⁶ they may not be useful in patients with early-stage RP or maculopathy. Hence, more localized functional tests are required for monitoring disease progression in patients with the latter manifestation of CRB1, which is gaining recognition as an important differential diagnosis of inherited isolated cystic maculopathy. CRB1-associated maculopathy is typically associated with a 9-bp deletion in exon 2,^9–11 with few exceptions.^7,8 Unlike CRB1-RP, patients with maculopathy can be myopic⁸ and have a well-developed retinal structure.⁷ The hallmark of CRB1-maculopathy in OCT is early-onset schitic/cystoid maculopathy, which resolves during several years to decades leading to severe macular atrophy.^10,11 However, none of the previous studies quantified the retinal sensitivity and macular volume parameters. In addition, there has been no report of a patient with biallelic CRB1 mutation with normal visual acuity and no apparent macular architectural change. 

In the present study, we use multimodal retinal imaging and microperimetry to characterize structural and functional changes in CRB1-associated LCA, RP, and maculopathy. In addition, we describe a previously unreported phenotype of asymptomatic fenestrated slit maculopathy. 

Patients and Methods

Subjects

The participants in this case series were enrolled as part of the Western Australia Retinal Degeneration (WARD) Study. Genetic diagnosis was established through the Australian Inherited Retinal Disease Registry and DNA Bank. The study protocol adhered to the tenets of the Declaration of Helsinki and ethics approval was obtained from the Human Ethics Committee of the Office of Research Enterprise, The University of Western Australia (RA/4/1/7916), and Sir Charles Gairdner Hospital Human Research Ethics Committee (approval number 2001–053). Written informed consent was obtained from the participants prior to inclusion in the study. 

Study Protocol and Clinical Assessment

Diagnosis of inherited retinal disease (IRD) was confirmed by an experienced retinal specialist (author F.K.C.) based on history and clinical examination and/or electroretinography (ERG). Clinical assessment involved obtaining a full medical and ocular history, best-corrected visual acuity (BCVA), and dilated fundus examination. ERG (RETIport version 3.2; Roland Consult, Brandenburg, Germany, or in-house custom built) was recorded in accordance with International Society for Clinical Electrophysiology of Vision (ISCEV) standards.¹⁷ Assessment of visual fields was undertaken using fundus-controlled microperimetry (Macular Integrity Assessment [MAIA] perimeter; CenterVue, Padova, Italy). The large 10-2 (68 test loci) and the small radial (37 test loci, 37R) grid patterns were used to map the retinal sensitivity profile within the macular (central 20 degrees field) and foveal (central 6 degrees field) regions, respectively (Supplementary Figs. S1A, S1B). 

Detailed multimodal imaging was performed in all cases, including widefield color fundus photography and green-light fundus autofluorescence (California; Optos plc., Dunfermline, UK) and spectral domain optical coherence tomography (SD-OCT, Spectralis; Heidelberg Engineering, Heidelberg, Germany). Fovea-centered macular volume scans were performed in a 30 degrees × 25 degrees area covering the early treatment diabetic retinopathy study (ETDRS) 1, 3, and 6 mm grid pattern (Supplementary Fig. S1C). Volume scans consisted of 61 horizontal B-scans separated by approximately 130 µm between each scan. Inner limiting membrane and Bruch's membrane were segmented automatically using the manufacturer software (HEYEX version 1.9.14.0; Heidelberg Engineering) and adjusted manually by a trained ophthalmologist (author D.R.) in cases with segmentation error. A dense raster 15 degrees × 10 degrees volume scan (97 slices, 30 µm intervals) was performed in cases with central residual island of vision to visualize the en face reflectance map of the interdigitating zone (IZ). Autofluorescence (AF) imaging was performed using a scanning laser ophthalmoscope (Spectralis HRA2; Heidelberg Engineering) with short-wavelength AF (SW-AF; λ = 488 nm) and near-infrared AF (NI-AF; λ = 787 nm) modalities. Flood-illumination AO retinal imaging using rtx1 AO camera (Imagine Eyes, Orsay, France) was performed in selected patients (n = 3) with fixation ability and without cataract and CMO. Multiple overlapping 4 degrees × 4 degrees images covering the central 6 degrees were taken at the photoreceptor level. Individual tiles were stitched together using i2k Retina software (DualAlign LLC, Clifton Park, NY, USA) and overlayed on high-resolution IR + OCT image. The location of the fovea was marked on the montaged AO image of each eye. 

To compare SD-OCT macular volume measurements in a normal population, 80 control subjects were grouped into three age categories: 20 to 40 years (n = 19), 41 to 60 years (n = 36), and 61 to 75 years (n = 25). Mean and standard deviations of macular volume indices were calculated for each age subgroup (Supplementary Table S1). 

Outcome Measures

BCVA was measured using the ETDRS chart and recorded as ETDRS letter score and converted to Snellen equivalents. Microperimetry point-wise sensitivity (PWS) was classified into normal sensitivity (≥25 dB), relative scotoma (0–24 dB), or absolute scotoma (<0 dB). Mean retinal sensitivity (MS), number of scotomatous loci and number of seeing loci (sensitivity ≥0 dB) were reported for both the small radial and 10-2 grids. 

The ETDRS grid (6 mm diameter) central subfield volume (CSV; 1 mm diameter), total macular volume (TMV), and average volume of the inner and outer ETDRS rings at four quadrants were recorded for each OCT volume scan. In addition, ratio of the TMV, outer ring volume (ORV) and inner ring volume (IRV) to CSV were calculated. AO images were analyzed using AODetect version 3.0 (Imagine Eyes). Cone density (CD) was measured at 2 degrees, 3 degrees, and 4 degrees along 4 meridional directions (Supplementary Fig. S1D) and angular values (cell/deg²) were reported. Patients’ CD values were compared with normal rtx1 ranges (average ± 2 SD) reported by Legras and colleagues.¹⁸ In addition, previously validated custom software¹⁹ was used for semi-automated segmentation of AO montages to create a parafoveal cone density map. 

Genotyping and Pathogenicity Assessment

Genomic DNA samples were collected, processed, and stored as previously described.²⁰ Genomic DNA was analyzed using the APEX ARRP microarray (Asper Biotech, Estonia), bidirectional Sanger sequencing (Macrogen, Seoul, South Korea, or Asper Biotech), or various next-generation sequencing (NGS) panels,²¹ and confirmed by Sanger sequencing (Casey Eye Institute, CEI Molecular Diagnostics Laboratory, Portland, OR, USA, or Molecular Vision Laboratory, Hillsboro, OR, USA). Cascade testing of family members was analyzed by Sanger sequencing, performed at CEI, the Australian Genome Research Facility (AGRF; Perth, WA, Australia), or at the Lions Eye Institute (Supplementary Table S2). 

Variants are described in accordance with Human Genome Variation Society recommendations.²² Pathogenicity was assessed using M-CAP,²³ REVEL,²⁴ Mutation Taster,²⁵ PolyPhen2,²⁶ SIFT, SIFT Indel,²⁷ VEST Indel,²⁸ and the Alamut Visual Splicing Suite, with information sought from LOVD, ClinVar, dbSNP, HGMD,²⁹ Exome Sequencing Project, and gnomAD.³⁰ Pathogenicity was interpreted in accordance with current guidelines proposed by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology,³¹ and associated literature.^32,33 

Statistical Analysis

Data were recorded in Statistical Package for the Social Sciences (SPSS) version 23 (SPSS/IBM, Inc., Chicago, IL, USA) and appropriate statistics applied after testing for normality. Quantitative data are presented as the mean (SD, range) and categorical data are presented as absolute numbers (%). For the macular volume parameters, the average and SD of the age-matched healthy subjects were calculated and values above or below 2 SD were considered significant. Z-scores of the patients’ macular volume measurements were calculated as ([patient value − normal average]/SD). Rates of annual change in macular volume parameters were calculated using linear regression analysis and beta estimates were reported. Only right eye data were included in all statistical analyses. 

Results

Patients

Twelve patients (5 male patients and 7 female patients) from nine Caucasian Australian families were recruited (Fig. 1). Patients were denoted by pedigree number and generation:individual. Mean age (SD, range) at baseline examination was 35 (16, 11–68) years. The mean (SD, range) age at onset of symptoms was 9 (7, 0–21) years. Demographics, clinical characteristics, and genotype are summarized in Table 1. 

Figure 1.

 

Pedigrees of families with CRB1-associated Leber congenital amaurosis (A), retinitis pigmentosa (B–E), and maculopathy (F–I). A novel phenotype (asymptomatic fenestrated slit maculopathy) was observed in two siblings (II:2 and II:4) of family 2647 I, whereas the other sibling (II:3) had typical macular dystrophy.

Table 1.

 

Demographics, Baseline Clinical Characteristics, and Genotypes of 12 Patients With CRB1-Associated Retinopathy

 

Spectrum of Phenotypes

Based on the pattern of retinal involvement on ultra-widefield FAF, patients were categorized into two groups, namely pan-retinopathy or maculopathy. Pan-retinopathy was further categorized into LCA and RP according to the age of onset and ocular findings. Maculopathy was categorized into macular dystrophy (MD) and asymptomatic fenestrated slit maculopathy (AFSM) based on symptoms and multimodal imaging findings. Using this classification, we had one patient with LCA, five patients with RP, four patients with MD, and two patients with AFSM (Fig. 2). Of note, patients 2647/II:3 and II:4 who were diagnosed with MD and AFSM, respectively, were di-zygotic twins. 

Figure 2.

 

Ultra-widefield color fundus photograph and autofluorescence (AF) imaging show diffuse nummular pigmentation and complete loss of normal AF in patient with Leber congenital amaurosis (A) and midperipheral bone-spicule pigmentation and hypo-autofluorescence with central preservation of retinal pigment epithelium in patients with retinitis pigmentosa (RP) phenotype (B–F). Note that preserved para-arteriolar RPE (PPRPE) is best visualized in AF imaging in all patients with RP B to F. Maculopathy presented with extensive macular atrophy incorporating the optic disc (G, H) with some residual temporal macular autofluorescence H, or localized superotemporal hypo-autofluorescence surrounded by hyperautofluorescent area (I), or normal fundus appearance and a macular hyperautofluorescent ring in AF (J) in patients with macular dystrophy, or unremarkable fundus and AF in two patients with asymptomatic fenestrated slit maculopathy (K, L). Mild loss of normal macular hyperautofluorescence and incomplete hyperautofluorescent ring may be noted in patient 2647/II:2 K. Note that patients 2647/II:3 J and 2647/II:4 L are di-zygotic twins with different presentations.

Central Retinal Function in Symptomatic Patients

Foveal and/or macular function was evaluated using microperimetry in 6 out of 10 symptomatic patients with severe outer retinal atrophy (3 RP and 3 MD). Serial microperimetry was available in 2 patients with RP (0432/II:4 and 2118/II:1) and 2 patients with MD (0625/II:1 and 0237/II:2; Fig. 3). Patient 0432/II:4, 48 years old, with RP, responded to 25/37 foveal stimuli and the foveal MS was 6.5 dB, whereas her BCVA was 20/250 (see Fig. 3A). After 4 years, the number of foveal seeing loci and foveal MS reduced to 20/37 loci and 4.9 dB, respectively (see Fig. 3C). The foveal sensitivity and number of seeing loci were 12.2 dB and 32/37, respectively, in the 29 year old patient with RP (see Fig. 3E). Patient 2118/II:1, 11 years old with RP, had a baseline macular seeing loci count and MS of 31 and 4.6 dB, respectively (see Fig. 3F). Follow-up examinations revealed persistent improvement in macular sensitivity over a 3-year period (see Figs. 3G, 3H). Patient 0625/II:1 with advanced MD (47 years old) had a BCVA of 20/125 in her better eye (right eye) and could see only 3 stimuli on foveal grid with a mean sensitivity of 0.2 dB at baseline, which remained stable after 4 years (see Figs. 3J–L). An unusual microperimetry pattern was observed in patient 0237/II:2 characterized by a localized superotemporal scotoma on the 10-2 grid. Follow-up examination after 3 and 4 years showed enlargement of the scotoma and significant decrease in macular MS (Figs. 3M–O). 

Figure 3.

 

Microperimetry showed residual foveal and/or macular function in patients with retinitis pigmentosa (RP) (A–H) and macular dystrophy (MD) (I–P). Central retinal function was recorded despite very low visual acuity (20/250) in the left eye of a 48 year old patient with advanced RP at baseline A and B, which declined after 4 years C and D. The 29 year old patient with RP showed greater foveal MS and number of seeing loci E. A fairly good baseline macular sensitivity with persistent improvement during a 3-year period was observed in the 11 year old patient with RP F to H. The 43 year old patient with advanced MD who presented with extensive macular atrophy responded only to stimuli presented at the central 2 degrees (I–J). Follow-up examinations showed stable function over a 4-year period (K–L). Patient 0237/II:2 presented with localized superotemporal scotoma in 10-2 test corresponding to the atrophic area, which progressed over 4 years of follow-up (M–O). The youngest patient with MD responded to all 68 presented 10-2 test loci with mild central depression (P). MS, mean sensitivity; SL, number of seeing loci. Color coding of threshold values is shown on the right side.

Only two patients with MD underwent ERG testing where patient 0237/II:2 showed a normal rod and subnormal cone response in full-field ERG and patient 2647/II:3 had features on multifocal and pattern ERG suggestive of macular dysfunction. 

Fundus Autofluorescence and SD-OCT in Symptomatic Patients

Ultra-widefield AF showed diffuse hypo-autofluorescence in patients with pan-retinopathy and highlighted PPRPE and a variable-sized central island of preserved retinal pigment epithelium (RPE) in all patients with RP phenotype, regardless of age and disease severity (see Figs. 2B–F and Supplementary Figs. S2A–E). SD-OCT over the PPRPE showed remnants of outer retinal and RPE structures blocking choroidal transmission of light (Supplementary Fig. S2F). Patients with MD revealed either extensive macular hypo-autofluorescence incorporating the optic disc in patients 0116/II:3 (68 years old) and 0625/II:1 (47 years old), localized superotemporal hypo-autofluorescence in patient 0237/II:2 (30 years old) or macular hyperautofluorescent ring in patient 2647/II:3 (23 years old; Figs. 2G–J). 

Macular SD-OCT showed coarse retinal lamination in patients with RP and diffuse outer retinal atrophy in all patients (Supplementary Fig. S3). Although remnants of photoreceptor layer were detected in all patients, an EZ line was visible only in 1 patient with RP and 3 patients with MD (Supplementary Figs. S3E, S3H, S3I, S3J). Bilateral cystoid maculopathy was observed in baseline OCT of patient 0237/II:2 and retrospective OCTs of patient 2647/II:3. Serial macular OCT revealed resolution of cystoid spaces and progressive outer retinal atrophy in both patients (Fig. 4). In addition, mild cystoid change was detected in three patients with RP phenotype (Supplementary Figs. S3B, S3C, S3F). 

Figure 4.

 

Natural history of cystoid maculopathy in two patients with macular dystrophy. Serial SD-OCT in patient 0237/II:2 (A–G) showing severe cystoid maculopathy at baseline A and gradual resolution of the intraretinal fluid B to D, followed by progressive outer retinal atrophy and perifoveal thickening D to G. Similar pattern was observed in patient 2647/II:3 as documented in retrospective time-domain OCT (H–K) and SD-OCT (L–N). Serial imaging in both patients revealed progressive attenuation and shortening of the ellipsoid zone (EZ). However, the span of the residual EZ was not measurable due to severe macular edema.

Macular Volume Profile

Baseline macular volume scan was available for 11 patients. None of the patients (including those with CMO in retrospective OCT scans) had cystoid maculopathy at the time of baseline imaging for the macular volume study and during follow-up phase. CSV was significantly lower than age-matched healthy controls in three of five patients with RP and three of four patients with MD, whereas TMV and ORV were significantly higher in four of five patients with RP and lower in two of four patients with MD. The ratio of TMV/CSV and ORV/CSV was higher than control group in four of five patients with RP and four of four patients with MD (8/9 symptomatic patients), with greater Z-scores for ORV/CSV (Table 2). All macular volume parameters were within normal ranges in the two patients with AFSM (see Table 2). 

Table 2.

 

Baseline Macular Volume Profile in Patients Compared With Age-Matched Healthy Controls

 

Longitudinal macular volume data were available in 10 patients (8 symptomatic and 2 asymptomatic) with a mean (SD, range) follow-up duration of 3.7 (1.9, 1.2–7.2) years. CSV declined at a rate of 0.002 to 0.004 mm³ per year in 5 patients (3 RP and 2 MD) aged 35 years or above and increased at a rate of 0.003 to 0.006 mm³ per year in 3 patients (2 RP and 1 MD) aged below 30 years. Conversely, TMV/CSV, IRV/CSV, and ORV/CSV increased in patients aged 35 years and above and declined in symptomatic patients aged below 30 years. This age-dependent pattern was not detected in TMV, IRV, and ORV change (Supplementary Table S3). Plotting volume measurements against age highlighted this age-dependent pattern, which was more prominent for TMV/CSV (Figs. 5A–C). Longitudinal change in OCT B-scan and macular volume profile in a 48 year old patient with RP with more than 7 years follow-up is shown in Figures 5D and 5E. Interestingly, despite significant decline in both CSV and TMV with increasing age, TMV/CSV ratio showed a significant increase (see Fig. 5, Supplementary Table S3). 

Figure 5.

 

Longitudinal changes in central subfield volume (CSV), total macular volume (TMV) and TMV/CSV ratio in eight patients (5 with retinitis pigmentosa and 3 with macular dystrophy) are shown in panels (A, B, and C), respectively. CSV increased in patients below 30 (n = 3) and declined in patients above 30 (n = 5) with increasing age A. TMV/CSV demonstrated a similar pattern, but in the opposite direction C. An example of cross-section B-scan of patient 0432/II:4 at baseline and after 8 years follow up is presented (D), beside volume and thickness change map (E). Central 1, 3, and 6 mm boundaries on B-scans are marked using yellow, blue, and green vertical lines, respectively. Despite notable decline in CSV (−0.03 mm³) and TMV (−0.86 mm³), the ratio of TMV/CSV increased by 6.02 E.

Multimodal Imaging in AFSM

Both patients with AFSM had normal BCVA, fundus appearance, and foveal sensitivity (Figs. 6A, 6F). However, NI-AF imaging revealed a horizontal linear hypo-autofluorescence marking (Figs. 6C, 6H) and corresponding attenuation of the subfoveal EZ and IZ on vertical dense raster SD-OCT scan in both patients, which was more evident on en face EZ map (Figs. 6D, 6E, 6I, 6J). AO cone density map showed moderate cone loss in the parafoveal region, which was more pronounced in the older sibling (Fig. 7A). Quantitative analysis of AO images showed low CD at 2 degrees eccentricity on all meridians in both patients with AFSM and 3 degrees and 4 degrees eccentricities on inferior and superior meridians in the older sibling (2647/II:2), as compared with normal ranges (Fig. 7B, Supplementary Table S4). 

Figure 6.

 

Microperimetry and multimodal imaging in the two patients with asymptomatic fenestrated slit maculopathy (AFSM). Both patients had 20/20 or better visual acuity and normal foveal sensitivity (A) and (F). Near-infrared autofluorescence (NI-AF) revealed a horizontal slit-like hypo-autofluorescent line (red arrows) in both patients (B, C and G, H). Vertical dense raster SD-OCT imaging through the hypo-autofluorescent lesion (green lines in panels C and H) demonstrated attenuated ellipsoid zone (EZ) and interdigitating zone (IZ) beneath the fovea (D and I, yellow arrows). Reconstructed en face EZ map highlighted the slit-like EZ defect (red arrows) in both patients (E and J). There is a minor vessel misalignment between EZ en face reconstructions and background infrared image in panel E, which is not expected to affect the morphology of the EZ defect. Normal OCT (K) and en face EZ map (L) using the same protocol in an age-matched healthy control is presented for comparison.

Figure 7.

 

Adaptive optics (AO) imaging findings in three siblings from family 2647 at same age range. Panel (A) shows cone density map and examples of cone mosaic imaging at 2 degrees eccentricity in patients with asymptomatic fenestrated slit maculopathy (AFSM) and macular dystrophy (MD) compared with a healthy subject. Density map showed moderate perifoveal cone loss in AFSM (second and third rows) and severe generalized cone loss in MD (bottom row). The color code in the bottom row shows cone density (× 10³ cells/deg²). Central 2.0 degrees (vertical) - 2.5 degrees (horizontal) is blocked due to lack of resolution to resolve foveal cones. Varying degrees of cone loss was observed in superior and inferior locations in AFSM and all locations in MD. Panel (B) shows cone density at 2 degrees, 3 degrees, and 4 degrees eccentricities in four meridional directions. The patient with MD had either no or few visible cones in most locations. Both patients with AFSM had lower cone density at 2 degrees location on all meridians. Additionally, patient II:2 had low cone density at all superior and inferior locations. The grey zone shows ±2 SD of normal CD values reported by Legras et al.¹⁸

Genetic Analysis

Genetic analyses identified 14 different CRB1 variants in this cohort (Supplementary Fig. S4), including 6 missense variants, 2 in-frame deletions, 2 nonsense variants, 2 splice site/near splice site variants, 1 frameshifting variant, and 1 substitution variant resulting in an in-frame exon deletion.³⁴ Of these, 11 were reported variants, including 1 hypomorphic variant, and the remaining 3 were novel. Of these, 13 were assessed as likely pathogenic or pathogenic and 1 was a novel variant of uncertain significance (Supplementary Table S5). 

Discussion

Pathogenic variants in CRB1 result in a wide spectrum of inherited retinal diseases ranging from LCA and adult-onset RP to maculopathy.^6,13 Accurate monitoring of disease progression has been hampered by the significant functional and structural damage already present at the time of diagnosis in most patients.¹⁶ Based on widefield FAF, we observed two broad CRB1 phenotypes: a pan-retinopathy and a maculopathy. We noted that all patients with RP had clearly visible PPRPE on widefield FAF. In both pan-retinopathy and maculopathy groups, we were able to quantify foveal sensitivity and the unique macular volume profile even in advanced stages of the disease. Furthermore, we report compound heterozygous mutations in CRB1 that resulted in both an MD and a previously undescribed phenotype of AFSM within the same family. We also report three novel mutations, including a splicing variant (c.652+5G>C) in one patient with MD, which is predicted to cause aberrant splicing and is likely pathogenic. 

Microperimetry: A Potential Functional End Point in CRB1-Retinopathy

Feasibility of microperimetry was previously shown in one patient with advanced CRB1-maculopathy using MP1 microperimeter (Nidek Technologies, Padova, Italy).⁷ The patient in this study was a 45-year-old woman who carried compound heterozygous CRB1 mutations, c.[3991C>T];[4142C>T]. Using 10-2 grid, the patient could respond to only 2 parafoveal stimuli in the better eye at baseline, which reduced to 1 stimulus after 5 years.⁷ We expand these findings to a larger sample of patients with CRB1 with varying disease phenotypes and severities and using the two MAIA grid patterns, and report a wide range of residual retinal sensitivities and progression patterns (see Fig. 3). We also demonstrate functional disease progression using both foveal (6 degrees) and macular (10-2) grids, although stable or improving function were also detected. For instance, we observed increasing MS and number of seeing loci in our 11-year-old patient with RP (Figs. 3G, 3H), which may be explained by lower sensitivity and higher variability of MAIA microperimetry in children.³⁵ Taken altogether, we suggest that microperimetry should be considered as a feasible method for documenting and monitoring residual retinal function in patients with CRB1-retinopathy. 

Ultra-Widefield FAF Enhances Visualization of PPRPE in all Patients With CRB1-RP

Although PPRPE in RP was described 4 decades ago for the first time,³⁶ its pathophysiologic mechanisms and clinical relevance are yet to be explored. It has been proposed that release of a permeable factor from retinal arterioles prevents degeneration of surrounding structures.³⁶ Whereas PPRPE was initially linked to CRB1,³⁷ genetic analysis may fail to detect a mutation in CRB1 in up to one third of patients with PPRPE.^37,38 In addition, there are reports of CRB1-RP without PPRPE,³⁹ indicating that PPRPE is neither pathognomonic nor mandatory for diagnosing CRB1-RP. 

The pan-retinal phenotype demonstrated bilateral PPRPE on ultra-widefield FAF in all our patients with RP. This feature was less prominent on color fundus photographs, particularly in those with less advanced disease. Studies that reported CRB1-RP without PPRPE either relied on fundus examination alone^15,39 or included patients with LCA.^14,40 Moreover, no other gene has been linked to RP with PPRPE so far, indicating that a thorough genetic analysis may unveil CRB1 mutation. Further studies are required to investigate the sensitivity and specificity of PPRPE for CRB1 mutation. However, given the finding that PPRPE can increase the likelihood of CRB1 mutations to up to 74%,^3,38 we recommend the use of ultra-widefield FAF imaging in all patients diagnosed with autosomal recessive RP to look for this specific sign. 

Perifoveal-to-Foveal Volume Ratio: A Potential Structural End Point in CRB1-Retinopathy

Perifoveal retinal thickening has been one of the most consistent findings in CRB1-associated retinopathies.^14,15,41 Jacobson and colleagues reported significant perifoveal retinal thickening in eight patients with CRB1-LCA/early-onset RP, whereas the retina was thinner compared with normal controls in two patients with RPE65 and one patient with GUCY2D. They used time-domain OCT (Zeiss Humphrey Instruments, Dublin, CA, USA) to measure cross-sectional retinal thickness in a single B-scan.¹⁴ Using time-domain Topcon 3D OCT-1000 (Topcon Medical Systems, Tokyo, Japan), perifoveal retinal thickening was observed in 77% (10/13)⁴¹ and 82% (9/11)¹⁵ of patients with CRB1-RP. In addition, increased central subfield retinal thickness (CRT) was noted in 36% (4/11)¹⁵ and 62% (8/13) of patients.⁴¹ Both CRT and perifoveal thickness showed an age-dependent decrease, which was attributed to resolution of macular edema or development of atrophy in the outer retinal layers with increasing age.⁴¹ Unlike all of the aforementioned studies that report time-domain OCT thickness results in LCA/RP, we report for the first time the detailed macular volume profile using the higher resolution SD-OCT in patients with both RP and maculopathy. We also introduce novel OCT parameters, TMV/CSV, and ORV/CSV that are increased in 89% (8/9) of patients with symptomatic CRB1-retinopathy. We propose that perifoveal-to-foveal volume ratio highlights relative foveal thinning and perifoveal inner retinal thickening, even if both values are within normal ranges. However, the implications of this marker in genotype-phenotype correlation remain to be explored in larger patient cohorts, including patients with other genetic diagnoses. 

Longitudinal analysis of macular volume parameters revealed a biphasic pattern in CSV and TMV/CSV, but not in TMV and ORV. None of our patients had significant macular edema at the time of baseline imaging for the macular volume study and during follow-up phase. Hence, in contrast to previous reports of macular thickness change with increasing age,⁴¹ it is unlikely that our observation was confounded by macular edema. We hypothesize that continued foveal gliosis after initial resolution of CMO may be responsible for increasing CSV until the early to mid-30s. After this phase, progressive foveal thinning results in decreasing CSV and increasing TMV/CSV and ORV/CSV with increasing age. Hence, CSV and TMV/CSV (but not TMV or ORV) might be considered as surrogate structural end points in CRB1 clinical trials, especially in late-stage disease. However, these speculations need to be investigated in larger populations with longer follow-ups and be correlated with histology. 

Asymptomatic Fenestrated Slit Maculopathy: A Novel CRB1-Maculopathy Phenotype

We detected the common c.498_506del mutation in 3 out of 4 families. This is an in-frame deletion in exon 2, which was classified as a hypomorphic allele and was reported most frequently in association with CRB1-associated maculopathy,¹⁰ as well as in 6 of 43 CRB1-associated LCA/RP families, including 1 homozygous and 5 heterozygous.³ The remaining family (0625) was diagnosed with compound heterozygous mutations, including the novel c.652+5G>C mutation, which is predicted to cause aberrant splicing and is likely pathogenic, and the previously reported c.2843G>A, which is a pathogenic missense mutation.¹³ Regardless of causative mutation, the typical natural history of CRB1-associated MD comprises childhood or juvenile-onset cystoid maculopathy followed by progressive macular atrophy and vision loss.^7–11 Cystoid maculopathy can develop at ages as early as 8 years old¹⁰ to as late as mid-40s,¹¹ and central atrophy can occur between the second⁹ and the fifth¹¹ decades of life. Clinical presentation of our symptomatic cases was comparable to those described in previous reports. In addition, we add long-term natural history of cystoid maculopathy using serial OCT in two patients (see Fig. 4), one of them (0237/II:2) reported as isolated foveal retinoschisis prior to genotyping.⁴² 

In addition to patients with typical CRB1-associated MD, we described 2 asymptomatic cases, who were siblings of a patient with typical MD, carrying biallelic c.498_506del and c.2843G>A variants with only subtle outer retinal changes. This novel AFSM phenotype, which was characterized by AO and microperimetry, has not been previously reported. In a series of 7 cases with the c.498_506del allele, 3 patients (including one homozygous) were asymptomatic at the time of presentation (10–30 years old), however, their visual acuity, OCT, and FAF were grossly abnormal.¹⁰ Conversely, our patients with AFSM displayed normal visual acuity and unremarkable OCT findings in the second decade of life. Long-term follow-up will unveil whether this new phenotype remains structurally stable (i.e. forme fruste state) or a preclinical stage that will evolve into MD as manifested by their sibling with the same variants. The clinical manifestation of AFSM may resemble occult macular dystrophy (OMD), a hereditary macular dystrophy characterized by foveal dysfunction despite normal fundus appearance. The most common genetic abnormality in OMD families has been the RP1L1 mutation with autosomal dominant inheritance. Furthermore, patients with OMD usually present with reduced visual acuity and central macular dysfunction,^43,44 which are not features found in our AFSM cases. 

Limitations

Our study was limited by a lack of ERG data. This would be useful in those patients with asymptomatic maculopathy. A baseline multifocal and pattern ERG could be helpful for detecting subclinical functional abnormalities in patients with normal visual acuity and foveal sensitivity. In addition, we have not provided long-term data for the novel AFSM phenotype described. 

Conclusions

We showed that PPRPE is best visualized with ultra-widefield FAF imaging in patients with CRB1-RP. Microperimetry may be useful in monitoring foveal function in moderate macular dystrophy cases given our observation of preserved foveal sensitivity. We found the macular volume profile, especially the ratio of perifoveal-to-foveal retinal volume, may be used as a surrogate structural outcome measure in both pan-retinopathy and maculopathy phenotypes even in the advanced cases. AFSM may represent a forme fruste state or preclinical stage of CRB1-associated MD. Longer term studies are required to validate the utility of microperimetry and macular volume profile in measuring disease progression rate. Recognition of AFSM phenotype provides an opportunity for examining disease modifying genes or early intervention to prevent cystoid maculopathy. 

Acknowledgments

The authors thank Amanda Scurry and Jayme Glynn for their assistance in organizing the patient appointments for the clinical assessments. The AIRDR acknowledges the assistance of Ling Hoffman and Isabella Urwin from the Department of Medical Technology and Physics at Sir Charles Gairdner Hospital. 

Supported by the Australian National Health and Medical Research Council under GNT116360 (to F.K.C.), GNT1188694 (to F.K.C.), GNT1054712 (to F.K.C.), and MRF1142962 (to F.K.C.), McCusker Foundation (to F.K.C.), Miocevich Retina Fellowship (to R.H.J.), Retina Australia (to J.A.T., T.L., J.N.D.R., and T.L.M.) and the UWA Postgraduate Research Scholarship (to D.R.). The sponsor or funding organization had no role in the design or conduct of this research. 

Disclosure: D. Roshandel, None; J.A. Thompson, None; R.C. Heath Jeffery, None; D.M. Sampson, None; E. Chelva, None; T.L. McLaren, None; T.M. Lamey, None; J.N. De Roach, None; S.R. Durkin, None; F.K. Chen, None