Special Issue Visual Function and Central Retinal Structure in

Elise Heon,1,2 Talal Alabduljalil,1 David B. McGuigan III,3 Artur V. Cideciyan,3 Shuning Li,2 Shiyi Chen,4 and Samuel G. Jacobson3

1Department of Ophthalmology and Vision Sciences, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada 2Program of Genetics and Genomic Biology, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada 3Department of Ophthalmology, Scheie Eye Institute, Perlman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States 4Clinical Research Services, The Hospital for Sick Children, Toronto, Ontario, Canada

Correspondence: Elise Heon, De- PURPOSE. To define the clinical phenotype of a cohort of patients affected with choroideremia. partment of Ophthalmology and Vision Sciences, The Hospital for METHODS. A retrospective study of patients with choroideremia included two centers. Data Sick Children, University of Toronto, collected included age, , , color vision, kinetic perimetry, optical Toronto, Ontario, Canada; coherence tomography (OCT), and genotype information. [email protected]. RESULTS. Sixty male participants were recruited. Genotype information was available for 58 Submitted: October 20, 2015 cases, and nonsense mutations were most commonly observed. Eight novel mutations were Accepted: December 16, 2015 identified including a missense mutation. The mean age at the first visit was 30.1 years (range, Citation: Heon E, Alabduljalil T, 5–65 years) and thirty-seven patients (61%) had more than one visit with a mean follow-up McGuigan DB III, et al. Visual function period of 10.3 years (range, 1–23 years). Visual acuity was not associated with age for patients and central retinal structure in cho- younger than 30 years (P ¼ 0.46) but significantly associated with age for the age group above roideremia. Invest Ophthalmol Vis 30 years (P < 0.0001). Central retinal thickness was significantly associated with visual acuity Sci. 2016;57:OCT377–OCT387. (P ¼ 0.03) and with age (P ¼ 0.0014). The extent of visual field documented by kinetic DOI:10.1167/iovs.15-18421 perimetry showed a negative correlation with age to tested stimuli; the smallest target used (I- 4e) showed the earliest and most rapid deterioration below the age of 20 years (P ¼ 0.0032). Color vision was abnormal in 46.7% of cases (mean age, 36.3 years; range, 18–61 years), which was associated with older age (P ¼ 0.0039). Central OCT images were abnormal in all cases, as early as age 10 years. Outer retinal tubulations were observed in all but five patients. No genotype–phenotype correlation was observed.

CONCLUSIONS. This comprehensive structural and functional characterization of a large cohort of patients with molecularly confirmed choroideremia indicates that certain parameters are not changing significantly with time while others are. The latter warrants a prospective natural history study, ultimately to be considered as outcome measures for interventional clinical trials. Keywords: choroideremia, blindness, OCT, photoreceptor, visual field, gene

horoideremia (CHM) is a monogenic X-linked (Xq21) choosing the right outcome measures in order to understand C condition characterized by progressive night blindness better the short- and long-term results of trialed interventions. and constriction of visual fields due to mutations in the CHM As clinical trials of gene therapy for CHM have been (REP1) gene.1–3 The progressive degeneration of the photore- initiated,9,11–17 a better understanding of the structural and ceptors, retinal pigment epithelium (RPE), and leads to functional changing features of the CHM- is needed. In severe peripheral field loss by adulthood and central visual the present study, a large cohort of men affected with acuity loss after 50 years of age.4 It is unclear whether the molecularly proven CHM was characterized with noninvasive primary insult is at the level of the choroid and RPE or the tests, including cross-sectional data and serial measurements, to photoreceptors. Our studies in patients support a primary gain insight into the disease’s natural history. photoreceptor disease, but analysis of a CHM mouse model suggests that mutations of CHM affect the integrity of 5–7 photoreceptors and RPE independently. The degenerative METHODS process of CHM is progressive, irreversible, and not treatable at this time. This was a retrospective study involving two sites: the Hospital Recent success in retinal gene transfer (GT) therapy has for Sick Children in Toronto (site 1) and the Scheie Eye Institute generated much enthusiasm and hope to make conditions such in Philadelphia (site 2). This work was approved by our as CHM treatable.8–10 Lessons learned from the early GT Institutional Ethics Review Boards and respected the tenets of studies8 highlight the important role of patient selection and the Declaration of Helsinki. Patients were identified through

iovs.arvojournals.org j ISSN: 1552-5783 OCT377

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TABLE 1. General Characteristics of the Populations Studied

Mean Standard Deviation Range Median IQR P Value

Age 30.17 16.39 5, 65 31.5 16.5, 43.0 0.54 Site 1, n ¼ 27 30.19 15.44 5, 65 32 18, 38 Site 2, n ¼ 33 30.15 17.37 6, 58 31 15, 46 Visual acuity 0.09 0.13 0, 0.65 0 0, 0.18 0.35 Site 1, n ¼ 27 0.06 0.09 0, 0.30 0 0, 0.10 Site 2, n ¼ 32 0.11 0.16 0, 0.65 0 0, 0.19 Interquartile range: the values are 25th percentile and 75th percentile, respectively. Mean age at first visit is 30.17 (SD ¼ 16.39) years. The two sites do not differ in terms of age (P ¼ 0.54). Median visual acuity is 0 (IQR ¼ 0–0.18). The two sites do not differ in terms of visual acuity either (P ¼ 0.35). IQR, interquartile range.

our respective internal databases. Inclusion criteria were (1) Statistical Analysis unequivocal clinical diagnosis of CHM, (2) molecular diagnosis of CHM when possible, and (3) availability of phenotype Statistical analyses were completed by using SAS 9.4 (SAS information (visual acuity, Goldmann kinetic visual field [GVF], Institute, Inc., Cary, NC, USA). color vision, and spectral-domain optical coherence tomogra- For descriptive statistics, a two sample t-test was used to compare patient age in the two sites. For visual acuity we only phy [SD-OCT]) for at least one visit. included the vision of the better eye and because visual acuity Data collected included deidentified demographic informa- was not normally distributed, a Wilcoxon two-sample test was tion, best-corrected visual acuity (BCVA) in logMAR,18 color used to compare patient visual acuity. We analyzed the vision (Farnsworth D15 panel), GVF, and OCT. Any color vision association between visual acuity and age by using linear anomaly was defined as abnormal. At site 1, OCT was spline mixed model. This model took into account the performed with the Cirrus (Carl Zeiss Meditec, Dublin, CA, correlation within a subject over repeated measures and USA), while site 2 used the RTVue-100 (Optovue, Inc., examined association between age and visual acuity for Fremont, CA, USA). At both sites, the OCT quantitative analysis younger patients (<30 years) and older patients (‡30 years) focused on the central retinal area (central subfield 1 mm) as separately. defined by the Early Treatment Diabetic Repeated measure ANOVA (RM-ANOVA) was used to 19–21 Study. At both sites, normal values were obtained from investigate the association between age and VF for each individuals with normal vision without ocular pathology, by stimulus (V-4e, IV-4e, III-4e, and I-4e). These RM-ANOVA measuring the central subfield thickness, from the Bruch’s models accounted for the doubly repeated measures in VF membrane to the vitreoretinal interface.19 Site 1 had 22 normal over time on the same subject and in VF tested on both eyes on individuals (mean age: 30 6 23.6 years) and site 2 measured 35 the same subject at each visit. A two-piecewise RM-ANOVA normal individuals (mean age: 33.1 6 15.1 years). The data model was built for stimuli I-4e. Normality of residuals of the were averaged at each site (site 1: 259 lm 6 22.37 lm; site 2 four models were checked and met. Bonferroni correction mean: 260.07 lm 6 23.37 lm). In cases and controls, only technique was used to adjust for the effect of multiple testing. scans with a clear foveal dip were included to measure central Furthermore, logistic regression was used to examine the retinal thickness (CRT). association between outcome color vision and predictors In a subset of patients, data from en face imaging with a including age and visual acuity. Mixed models were used to confocal scanning laser ophthalmoscope (Spectralis HRA; examine the association between outcome CRT and predictors, Heidelberg Engineering, Heidelberg, Germany) using near- including acuity and age, separately. infrared (815 nm) illumination was analyzed. Regions of relatively low reflectance within the macula on these images represent the remaining extent of melanized RPE.22 A RESULTS nonlinear gamma transformation (power ¼ 0.7) was applied Sixty male CHM patients were included in this study. Thirty- to gray levels in order to allow simultaneous visualization of seven patients (61%) had more than one visit with a mean features both within darker and brighter regions of the images. follow-up period of 10.3 years (range, 1–23 years). The data of The GVF data were presented in percentage of residual field patients at both sites were similar (Table 1) and analyzed of vision compared to age-matched controls for specific together. Although we could not capture age at diagnosis, the 23 isopters. The visual field (VF) areas were quantified by using mean age at the first visit was 30.1 years (range, 5–65 years; 24 a previously published computer-based algorithm. Test Table 1). points of the Goldmann field were digitized and mapped as points on a VF hemisphere. Their sequence defined a The Correlation With Age Differs Between Visual curvilinear polygon, and the solid angle (in steradians) subtended by it was calculated. The entire field of vision was Fields and Visual Acuity accounted for as the solid angles associated with The mean refractive error in spherical equivalent was 2.75 D were subtracted from the seeing areas. For easier interpreta- 6 3.5 D. Although BCVA appeared to decrease with age (Table tion, solid angle measurements are reported as a percentage of 2; Fig.1), a beta regression analysis of the visual acuity over the mean normal ‘‘visual field extent’’ for the corresponding time did not show any significant relationship between age and target. The annual rate of change for V-4e and I-4e test targets visual acuity (P ¼ 0.63). However, although there was no was calculated from a subset of patients with serial data as correlation between BCVA and age when younger than 30 previously described.25,26 Both sites had a practice of adapting years (b ¼0.00174, P ¼ 0.46), for patients older than 30 years, the patient to the perimeter environment for approximately 5 visual acuity declined significantly as age increased (b ¼ minutes. 0.008871, P < 0.0001). The estimated visual acuity between

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TABLE 2. Summary of Data Goldmann Visual Field

Age, VA, Color V4e, IV4e, III4e, I4e, III3e, Central Subject y logMAR Mutation V OD/OS OD/OS OD/OS OD/OS OD/OS OCT (ORT)

1 40 0 p.F227Lfs*5 Normal 62.5/88.5 NA 2 27 0 c.1349þ2insGGT Normal NA NA NA NA NA NA 3 54 0.88 p.A455Qfs*3 Abnormal 0.1/0.1 1 4 20 0.1 p.C406* Normal 6.1/6.6 2 5a 65 0.18 p.L407H Abnormal 0.2/0.08 1 6 47 0.6 c.940þ1G > T Abnormal 2 7 35 0 p.I243Lfs*4 Normal 60.2/67.5 14.2/13.8 2 8 32 0.1 p.A455Qfs*3 Normal 16.8/13.5 3/0.7 0.2/0.2 2 9b 28 0 p.K415* Abnormal 66.1/77.3 NA 10b 42 0 p.K415* Normal 47.8/58.2 NA 11 33 0.1 del exon 10-12 Normal 38.6/30.8 NA 12 65 0.3 p.C406* Abnormal 6.7/17.4 1 13 51 0.3 p.C416* NA –/0.5 0 14 27 0.1 p.C409* Abnormal 4.8/8.7 NA 15 27 0 del exon 3-4,6-12 Normal 88.7/80.7 2.7/0.9 NA 16 38 0 del exon 3-8 Abnormal 22.7/24.8 NA 17 57 0.6 c.49þ3A > G Abnormal 0.5/0.5 0.2/0.3 2 18 41 0.1 p.R270* Normal 4.1/6.1 0.7/0.8 2 19 41 0.1 p.R239* Normal 65.6/66.3 2.6/2.4 2 20 62 0.18 p.E179* Abnormal NA 21 24 0.3 p.R253* Abnormal 34.5/41.4 3.4/1.5 2 22 15 0 p.Y565* Normal 98.4/102.6 87.7/92.8 NA 23a 25 0 p.L407H Abnormal 89.5/90.9 49.4/92.8 2 24 45 0.18 p.Y565* Abnormal 0.9/1.5 2 25c 14 0 p.S445* Normal 27.9/40.1 0 26c 11 0 p.S445* Normal 113.3/107 90.3/81.6 33.6/31.7 1 27 19 0 c.49þ3A > G Normal NA NA NA NA NA 2 28 51 0.2 p.R270* Abnormal 0.4/0.2 0.02/0 0 29 18 0 del exons 3-9 Abnormal 111.8/104.3 1.4/2.3 2 30 61 0 p.R270* Abnormal 0.3/0.2 0.1/0.02 NA 31 38 0.18 p.R555* Normal 22.6/18.1 0/0.2 NA 32d 43 0.1 p.D183Efs*14 Abnormal 3.4/5.3 0.2/0.2 2 33 37 0 p.R267* Normal 89.8/68.3 0.5/0.3 2 34d 13 0.1 p.D183Efs*14 Normal 98.1/89.7 0.9/0.9 2 35e 14 0 p.R253* Normal 98.4/85.4 6.3/3.6 1 36e 9 0 p.R253* Normal 99.5/99.3 10.3/19.3 0 37 58 0.7 del exon 9 Abnormal 3.6/– 0/– 1 38 13 0.1 p.R253* Normal 86.1/92.3 1.5/2.5 NA 39 25 0.1 c.1244þ1G> A Normal 93.7/84.8 0.9/0.9 2 40 17 0.1 p.E542Kfs*13 Normal 56.6/53.7 0.5/0.3 2 41 58 0.2 p.R253* Abnormal 5.8/7.4 0.04/0.04 2 42 44 0 del exon 1 Normal 42/45.9 0.08/0.2 0 43 24 0.1 NA Normal 28.2/36.9 0.07/0.2 NA 44f 37 0 p.R267* Normal 48.4/53.7 0.5/0.4 2 45f 47 0.1 p.R267* Normal 15.7/12.7 0.2/0.1 1 46g 41 0 c.116þ1G> A Abnormal 71.6/75.2 0.2/0.1 1 47 56 LP p.E177Kfs*20 Abnormal –/– –/– NA 48 46 0.4 p.V529fs*7 Abnormal NA NA NA NA NA NA 49 50 0 p.R293* Normal NA NA NA NA NA 1 50 26 0 NA Abnormal NA NA NA NA NA NA 51g 39 0 c.116þ1G> A Normal NA NA NA NA NA NA 52 12 0.3 del exons 9-11 Abnormal NA NA NA NA NA 1 53 32 0 c.1166þ1G > A Abnormal NA NA NA NA NA 2 54 57 0.3 p.R267* Abnormal NA NA NA NA NA 1 55 23 0.1 p.Q380* Normal NA NA NA NA NA 1 56 53 0 p.S28Qfs*32 Normal NA NA NA NA NA 2 57e 58 0.3 p.R253* Abnormal NA NA NA NA NA NA 58e 56 0.1 p.R253* Abnormal NA NA NA NA NA 1 59 39 0.3 c.1350-14_1350-10del Normal NA NA NA NA NA 2 60 6 0.3 p.Q380* Normal NA NA NA NA NA NA Subject numbers with a letter refer to the family they belong to. The number in the last column refers to the number of eyes involved. Age, age at test; Color V, color vision testing; LP, light perception; NA, not available; OD, right eye; OS, left eye; VA, visual acuity.

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the V-4e (P < 0.0001) and III-4e (P ¼ 0.018) isopters was also significant. The pattern of visual field loss was symmetric and independent of the class of the mutation (nonsense, frameshift, or missense). Practically, by 20 years of age there was approximately 1% of the I-4e field left, while 50% of the V-4e field was maintained until reaching the 40s. In analysis 2, we studied a subset of 17 patients with available longitudinal data (n ¼ 10 for V-4e; n ¼ 9 for I-4e) and assumed a model wherein the disease progresses exponentially after its onset.27–32 The estimated rates of visual field constriction were 8.3% (64.7%) per year for the large target and 27.7% (619.2%) per year for the small target (Figs. 2C, 2D). Color vision results were available for 60 cases (Table 2, Fig. 3) of which 28 were abnormal (46.7%); 17.5% of this subgroup had normal visual acuity (mean age, 36.3 years; range, 18–61 years). Five patients had no measurable color vision despite a BCVA of 0.3 or better (Table 2; cases 12, 14, 20, 24). Visual acuity was not associated with abnormal color vision (P ¼ 0.11) but older age was (P ¼ 0.0039). Mean age of patients with abnormal color vision was 43 years (range, 12–65 years) while that of cases with normal color vision was 27 years (range, 6– 53 years). Each additional year increased the risk of developing abnormal color vision by 6% (OR ¼ 1.06, 95% CI ¼ 1.02–1.11).

Optical Coherence Tomography: Early Central FIGURE 1. Best-corrected visual acuity in CHM. The better visual acuity of each patient (n ¼ 60) is shown at age of testing. When serial data are Retinal Changes Despite Good Vision available (n ¼ 37), visual acuities from the same eye at the first and the latest evaluation are plotted and connected by lines. Thicker line The OCT images were available for 39 cases (61 eyes). The CRT represents the linear spline mixed model (fit by excluding the datum was within normal limits until the 40s, followed by significant from the patient with hand-motion acuity) described in text. thinning in some patients between 40 and 60 years of age, while others maintained normal thickness in this age group (Fig. 4). Mixed-model analyses of the mean CRT (259.2 lm 6 birth and 30 years was not significantly different from normal. 52.70 lm) showed an association with age (P ¼ 0.0045) as well However, the estimated vision at 50 years of age was as an association with visual acuity: the older the patient, the significantly worse at 0.21 (P < 0.0001). There was no loss thinner the CRT (b ¼1.73, P ¼ 0.0014) and the worse the eye of light perception in this cohort. vision, the thinner the CRT (b ¼22.68, P ¼ 0.0321). Goldmann kinetic visual fields were measurable for 57 Paramacular retinal disorganization was also a significant individuals. Examples of the different patterns of progressive feature observed early (Fig. 5A). Outer retinal tubulations visual field loss of four representative patients are shown (Fig. (ORTs) were observed in all but five patients (Tables 2, 3; Fig. 2A); P29 lost most of his I-4e field throughout 7 years (ages 11– 5). Thirteen patients had ORTs in one eye, while in 22 cases 18 years), while fields to the V-4e target remained relatively full ORTs were observed in both eyes. The presence or absence of during that period. P43 had a very small field to the I-4e target ORTs did not correlate with age or visual acuity. We used en at age 17 years and this decreased further throughout 7 years. face OCT imaging to determine the branching patterns of the The midperipheral scotomas (to the V-4e target) also expanded ORTs as has been demonstrated in age-related macular during the 7-year period depicted (ages 17–24 years). P42 at degeneration (AMD).32–36 Two patients, one at age 12 years age 31 years had a very small field to the I-4e target, which got and the other at age 37 years, showed differently sized central smaller during the 12-year period. He also showed an islands of RPE on near-infrared reflectance images (Figs. 5B, altitudinal (superior) loss of visual field (to the V-4e target) 5C). En face and cross-sectional imaging of the central, and the beginning of an inferior scotomatous region in the near normally pigmented region is also shown (right). Both patients midperiphery. During the subsequent 12 years, the showed branching structures evident at the edges of the encircled fixation and by age 43 years, the central island was preserved RPE and extending to further eccentricities. Insets isolated from a residual far temporal peripheral island. P28—at adjacent to the en face images (Fig. 5) are drawings of the a more advanced stage for his age than the other patients branching patterns. Although this warrants further investiga- shown—only had a small central island and peripheral inferior tion, the caliber of the tubular structures seemed to be wider in temporal islands of perception with the V-4e target at age 33 the younger patient. An OCT cross-section at one location in years. The peripheral islands became nondetectable at age 51 each patient showed correspondence between the branched years but the central island remained detectable (<58) with the structures and the ORTs. I-4e target and further decreased in size at the latest age. In our cohort only one eye displayed cystoid macular Visual field extent from the entire cohort of patients, using a edema. However, in 20% of eyes imaged there were mild number of different stimuli (Table 2), is plotted in Figure 2B. degenerative cystic changes in the inner retina, unrelated to The field of vision to the I-4e stimulus showed the earliest (first age, suggestive of a schisis (Fig. 5A). The OCT imaging of the decade) and most rapid decline in extent, while the fields in central retina also showed other previously described fea- response to the V-4e stimulus could be measured ~30 years tures,7,37,38 such as interlaminar bridges, loss of the outer longer. Two types of analyses were used to interpret the visual retina, cystic spaces, focal RPE hyperplasia, all of which were fields changes. Analysis 1 based on the two-piecewise RM- independent of each other. The thinning of RPE/Bruch’s ANOVA model showed that the visual field area loss correlated membrane complex increased with age but could still be seen with age for the I-4e isopter for patients 20 years or younger (P in some individuals 60 years of age. Although we did not ¼ 0.0032). The correlation of visual field area loss with age for measure the choroidal thickness, paramacular choroidal

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FIGURE 2. Kinetic visual fields in CHM. (A) Serial visual field drawings (using the V-4e and I-4e stimuli) from four patients, representing different disease stages at different time points, illustrate progression of visual field loss in CHM. (B) Quantified field extents to five different targets are plotted on a linear scale, with serial data connected by lines for cases with more than one measurement. Data points from the four selected patients in (A) are marked (P28, P29, P32, P43). (C, D) In a subset of patients with serial data, the annual rate of change was estimated for V-4e (C) and I-4e (D). Data were arranged as time after onset of decline in field extent; the annual rate of decline (dashed gray line) is the average of the slopes fit to the log-linear data for individuals.

thinning was obvious by late teens (P17) and slowly The Phenotype of Missense Mutations Is Not progressed centrally. Choroidal changes also included in- Different From That of Truncating Variants creased size of choroidal lacunae and progressive collapse. Mutations (n ¼ 37) were identified in 58 cases (Tables 2, 4), The choroid was barely perceptible by the age of 60 years. most of which were private. There were eight novel mutations 39 Choroidal excavation was noted in at least three eyes (P3, P5, including one missense mutation in two distantly related and P12) at ages 54, 64, and 65 years, the significance of which individuals, which is rarely documented. The phenotype of has not been determined (Table 2). individuals with the missense mutation did not appear different

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FIGURE 3. Color vision defects can occur early and progress with age. The horizontal axis reflects three age groups: 5 to 20 years, 20 to 35 years, and 35 to 65 years.

from that of CHM patients with other mutation types, but this followed by segregation analysis and determination of allele may reflect a limitation due to sample size. In fact, no frequency in a specific ethnic background if indicated. phenotype–genotype correlations were observed at large. As documented in previous studies, the nonsense mutations were most common (40%) followed by the frameshift, splice site, DISCUSSION and large deletion variants in equal proportion (19%). Novel mutations were validated, using information from the literature Extending Previous Observations About Vision in and various mutation databases (https://grenada.lumc.nl/ CHM LOVD2/Usher_montpellier/variants.php?select_db¼CHM& action¼view_unique; provided in the public domain by the There is a long history of clinical description of patients with Leiden Open Variation Database). The potential effect of novel the diagnosis of CHM as well as some reports that include variants was predicted by using Polyphen-240 and SIFT41 postmortem eye donor retina histopathology.42–46 Most of the

FIGURE 4. Central retinal thickness of CHM patients as a function of age; CRT in choroideremia. Scatter plot of CRT values from patients as a function of age from both sites (site 1 [pink] used the Cirrus OCT, while site 2 [blue] used the Optovue). Mean retinal thickness values by age were estimated by using local polynomial regression fitting. The shaded areas show the sample variation (61.96 standard deviations) of CRT normal values for each site. The retina appeared to be initially on the thicker end of normal followed by a slow thinning after the age of 50 years.

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FIGURE 5. Retinal structural features in CHM. (A) The SD-OCT scans along the horizontal meridian through the fovea are shown from six different patients. Black arrows show ORTs. White arrows show schisis in the ganglion cell layer. White-filled arrowheads of black arrows point to cystic lesions. (B, C) The NIR reflectance images are shown for comparison with an OCT en face image in 12-year-old and 37-year-old CHM patients. Retinal regions of low reflectance correspond to pigmented RPE, whereas those with high reflectance likely correspond to RPE atrophy. Thus, hyperreflective ORTs border the retained pigmented RPE and extend out into the atrophied area as small tubular structures. An OCT cross-section, selected from the many individual scans used to derive the en face image (white line indicates where section was taken), illustrates that the hyperreflective features correspond to ORTs. Insets: Diagram of central pigmented RPE (filled-in gray) with surrounding tubular structures (lines).

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TABLE 3. Summary of Outer Retinal Tubulation Correlation

ORT No. Cases Age, Mean (Range), y VA, Mean (Range), logMAR Mutation Type No. Mutations

None 5 33.8 (9–51) 0.1 Nonsense 3 Splice 0 fs 0 Del 1 Missense 0 1 eye 12 43.23 (12–65) 0.22 Nonsense 8 Splice 1 fs 1 Del 1 Missense 0 2 eyes 22 34.72 (13–58) 0.13 Nonsense 9 Splice 6 fs 5 Del 1 Missense 1 The number of eyes involved is indicated for the ORTs. Del, large deletion; fs, frameshift mutations; Splice, mutation involving a splice site.

original literature has preceded molecular confirmation but the Color vision was abnormal in half of the patients studied, as distinctive fundus appearance and X-linked inheritance make early as 12 years of age, and this correlated with age. Abnormal those reports relevant.47 The current work provided a color vision parallels the observation that the cone mosaic is comprehensive documentation of functional and OCT struc- disturbed on adaptive optics scanning laser ophthalmoscopy tural phenotype as a consequence of REP1 mutations, some of (AOSLO) imaging despite an often normal cone density and which are novel. Limitations of the current study included the normal central visual acuity.51 The youngest case to have retrospective nature of the data and the lack of regular serial abnormal color vision was 12 years of age (P52; visual acuity: follow-up intervals. Strengths included relatively large number 0.3 logMAR and GVF I4e: 0.5% of normal), while the oldest to of patients from two centers with overlapping approaches and maintain normal color vision was 53 years of age (P56; visual relatively long-term follow-up period. acuity: 0 logMAR). The color vision was often maintained until For patients with REP1 mutations, it is clear that the central the late 50s (Table 2; Fig. 3). Studies of color vision assessment visual acuity and central retinal thickness by OCT are usually in patients with CHM have yielded variable results with the well preserved until the fourth decade of life and sometimes most recent studies documenting some anomalies.52–55 Unlike longer. Although central visual acuity changes did not correlate 54 with age younger than 30 years (P ¼ 0.46), it did for those older the recent study of Jolly et al., we did not observe any than 30 years (P < 0.0001), therefore a measure of disease correlation with visual acuity (P ¼ 0.11) but saw a correlation 54 progression only for that age group. Because the visual acuity with age (P ¼ 0.003). Our work supports that of Jolly et al. in changes observed were not linear, we could not determine a that the color vision changes did not follow any specific axis. reliable time-related rate of vision loss unlike previous These early functional changes in color vision reflect early studies.48–50 It has been reported that 33% of patients older cone dysfunction, which provides insight in the CHM-related than 60 years have 20/200 or worse vision.49 In our cohort of degeneration and an additional potential outcome measure. patients older than 60 years (n ¼ 4), the average visual acuity The findings in the current work and that of Jolly et al.54 was 0.22 logMAR with one individual having normal vision. indicate that a standardized but practical and sensitive measure

TABLE 4. Summary of Mutations Identified by Mutation Type

Nonsense, Frameshift, Splice Site, Large Deletions, Missense, n ¼ 15 A n ¼ 7A n ¼ 7An ¼ 7An ¼ 1A

p.C406* 1 p.F227Lfs*5 1 c.1349þ2insGGT 1 Del ex 10-12 1 p.L407H 1 p.T756* 1 p.A455Qfs*3 1 c.940þ1G > T 1 Del ex 3-4,6-12 1 p.K415* 1 p.D183Efs*14 1 c.49þ3A > G 1 Del ex 3-8 1 p.C416* 1 p.E542Kfs*13 1 c.1244þ1G> A 1 Del ex 3-9 1 p.C409* 1 p.E177Kfs*20 1 c.116þ1G> A 1 Del ex 9 1 p.R270* 3 p.V529Hfs*7 1 c.1166þ1G > A1Del ex 1 1 p.R239* 1 p.S28Qfs*32 1 c.1350-14_1350-10del 1 Del ex 9-11 1 p.E179* 1 p.R253* 2 p.Y565*1 p.S445*1 p.R555* 1 p.R267* 3 p.R293* 1 p.Q380* 1 Underlined mutations are novel. A, number of unrelated alleles for which a mutation is seen; Del, deletion; ex, exon; n, number of unrelated alleles.

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of color vision should be included in the workups of patients like structures formed by abnormal photoreceptors4,46 and with CHM. these may be the ORTs seen with OCT. The rosette-like Kinetic perimetry has been a traditional outcome measure structures were documented in a postmortem retina of a 19- for peripheral visual field measurement and there have been year-old CHM patient,46 indicating that our observations of many examples of fields shown in CHM studies.42–45,47,56 To ORTs in younger patients has a morphologic basis. Our our knowledge, there has not been an attempt to date to comparison between OCT en face and cross-sections showed quantify and characterize the rate of change of perimetry with that the ORTs in CHM are also in the outer nuclear layer (ONL), age in CHM. We reviewed kinetic perimetry results of 57 which is similar to the well described histopathologic studies patients (33 had data from two or more visits) and showed that of ORTs in AMD.34,73 Whether the ORTs of CHM represent the changes observed for the I-4e, III-4e, and V-4e isopters tubes of degenerating cone photoreceptors, as in AMD,34,73 is were related to age. The changes observed with the I-4e isopter uncertain. In donor studies of RP, there have been rare reports were the earliest and the fastest (Fig. 2). This is consistent with of rosettes comprised of S-cones but not rods or L/M-cones74 most other such studies of RP using comparable meth- and rosettes comprised of mostly rods but not cones.75 ods.25,26,29,57–62 For the V-4e target, the CHM visual field Considering that the ORTs in CHM were adjacent to but not change of 8.3% per year is within the lower rates published by within preserved areas of ONL, it can be speculated that they previous pigmentosa studies, other than specifically are a sequela leading from photoreceptor degeneration, as an adRP (ranging from approximately 5%–10% per year).61,63,64 unusual form of retinal remodeling. The phases of retinal Usher syndrome studies have tended to report higher rates of remodeling have been studied in great detail for more than a decline per year for V-4e (ranging from 11%–14%28,29). The decade in many murine models of retinal degeneration and to rate of change related to the I-4e target, 27.7% per year, was our knowledge, there is no specific description of a structure higher than for the V-4e rate in this cohort of CHM patients and resembling an ORT.5,6,76 The pseudorosettes widely seen in results were generally similar, albeit with slightly faster mouse retinopathy models,77 as well as other types of rosettes, progression, to those reported for other forms of RP.28,58 have been considered different than ORTs.34 Further en face The kinetic perimetry data we presented allow for imaging, extending as far as the OCT will permit, should at prediction of field loss in CHM and will be useful for guiding least answer questions about the more peripheral extent of the patients inquiring about a prognosis. Measures of intervisit tubular structures and whether they stop or continue beyond variability were not performed and this would be required if borders of more viable-appearing RPE and ONL. kinetic perimetry was considered for disease monitoring. Results of static threshold perimetry would be very valuable What Is the Clinical Relevance of the Current to monitor patients in clinical trials. Such data have not been reported for the CHM peripheral field, either as dark- or light- Results? adapted threshold perimetry. Ongoing GT trials of CHM are As gene augmentation clinical trials have started in mainly subretinal injections directed at the central retina.9 The CHM,6,9,13–15,17,78,79 it becomes important that molecular use of fundus perimetry (also called microperimetry) has been testing be available to any patient with a CHM-like phenotype very helpful to date to understand central retinal function and and that the CHM phenotype be thoroughly characterized. would be useful in assessing CHM patients. Future trials, Defining the phenotype as a function of time is important to whether GT or other treatment modalities, may target accurately stage the disease process and understand outcomes. extracentral retina in which case threshold data for change in From the ongoing gene therapy clinical trials, it is acknowl- peripheral function will then be required to decide the most edged that the initially selected participants were at different favorable treatment sites and to monitor posttreatment disease stages, which challenges the interpretation of results.10 changes. It is now apparent that central visual acuity is not a good parameter of the disease stage for CHM patients younger than Retinal Structure Following the Effects of REP1 30 years, while the OCT, color vision, and visual fields could Mutations provide more useful information. An understanding of the natural history of CHM-related retinal degeneration, the Retinal structural changes using OCT have been observed to characteristics of the phenotype, and the correlation between involve different layers of the retina in CHM patients.7,37,50,65,66 surrogate measures will assist in properly staging the disease Thinner CRT was significantly associated with worse visual severity. Accuracy in defining a ‘‘stage’’ of retinal degeneration, acuity and older age. An important observation was that ORTs similar to what is done for tumors,80,81 is becoming were observed in 34 of the 39 cases (87%) imaged; there was increasingly important to optimize the design and selection no obvious relation to age or the type of mutation involved. of interventions and candidates. Outer retinal tubulations have also been noted in a number of Phenotyping of CHM and other retinal dystrophies merits other inherited retinal degenerations,67–69 but only Bietti the development of some guidelines that would include the crystalline retinopathy seems to show ORTs frequently.70,71 structural and functional measures of retinal degeneration. This Other studies have reported ORTs in CHM,7,37,51,69,72 but the would allow a better interpretation of larger sets of data from high frequency of this pathologic finding in our cohort leads to different centers, and most likely better reflect the natural the suggestion that ORTs may be an expected part of the CHM history of disease as well as allow an optimal selection of phenotype. The presence of ORTs in patients at early ages outcome measures and patient management. suggests the effect is not simply a late-stage abnormality. Our en face OCT imaging technique, as has been used in studies investigating ORTs in AMD,33,35 revealed many branched Acknowledgments structures surrounding the area of retina with residual The authors are grateful for assistance in figure design by Thomas photoreceptors and RPE. Correlative OCT cross-sections Wright, PhD, Alejandro J. Roman, MSc, and Alexander Sumaroka, indicate that these branched structures are the ORTs, as has PhD. also been shown in AMD images.33,35,36 Supported by the Mira Godard Research Fund, the Brendan Eye What is the histopathologic basis of these features and how Research fund (EH), and Foundation Fighting Blindness (SGJ, AVC). do they relate to the pathophysiology of CHM? Human The authors alone are responsible for the content and writing of postmortem retinal histopathology in CHM has noted rosette- the paper.

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