Retina Blue Cone Caused by the C203R Missense Mutation or Large Deletion Mutations

Alexander Sumaroka,1 Alexandra V. Garafalo,1 Artur V. Cideciyan,1 Jason Charng,1 Alejandro J. Roman,1 Windy Choi,1 Supna Saxena,1 Valeryia Aksianiuk,1 Susanne Kohl,2 Bernd Wissinger,2 and Samuel G. Jacobson1 1Scheie Eye Institute, Department of , Perelman School of , University of Pennsylvania, Philadelphia, Pennsylvania, United States 2Molecular Genetics Laboratory, Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tuebingen, Tuebingen, Germany

Correspondence: Samuel G. Jacob- PURPOSE. To compare the phenotype of blue cone monochromacy (BCM) caused by large son, Scheie Eye Institute, University deletion mutations with those having the C203R missense mutation. of Pennsylvania, 51 N 39th Street, Philadelphia, PA 19104, USA; METHODS. BCM patients with large deletion mutations (n ¼ 21; age range, 5–60 years), and [email protected]. with the C203R missense mutation (n ¼ 13; age range, 5–70 years), were studied with optical coherence tomography, visual acuity, and perimetric sensitivity in a retrospective Submitted: July 15, 2018 Accepted: October 29, 2018 observational case series. Perceptual estimates of spatial resolution driven by rods, S-cones, and L/M-cones were obtained by the choice of chromatic gratings presented on varied Citation: Sumaroka A, Garafalo AV, adapting conditions with a modified microperimeter. Cideciyan AV, et al. Blue cone mono- chromacy caused by the C203R mis- RESULTS. Both genotypes had abnormal foveal photoreceptor structure early in life. Patients sense mutation or large deletion with the C203R mutation, however, had decades-longer persistence of foveal photoreceptor mutations. Invest Ophthalmol Vis Sci. outer nuclear layer thickness and a slower rate of development of inner segment/outer 2018;59:5762–5772. https://doi.org/ segment defects than did patients with large deletion mutations. At late ages, both genotypes 10.1167/iovs.18-25280 had comparably severe losses of central structure. At the rod-rich hot spot, there was no difference in structure between cohorts with age. Grating acuities in all BCM patients were driven by S-cones and rods; the foveal structural differences were not reflected in a difference between cohorts in visual sensitivity and spatial resolution.

CONCLUSIONS. A difference in structural phenotype due to the C203R mutation versus large deletion mutations in BCM was detected as a more prolonged persistence of foveal photoreceptor structure in patients with the missense mutation. This should be taken into account in planning natural history studies, selecting outcomes for clinical trials, and defining the time window for possible . Keywords: mutations, cones, optical coherence tomography, visual acuity

lue cone monochromacy (BCM) is an X-linked disease that The goal of the current study was to compare the B is characterized by impaired red (L, long wavelength) and phenotype of the other common BCM genotype—the C203R green (M, middle wavelength) cone photoreceptor function missense mutation—with that of the large deletion mutations. caused by mutations in the OPN1LW/OPN1MW gene cluster on The results indicate very different disease progressions in chromosome Xq28. Studies of the molecular basis of BCM have BCM due to the two most common types of mutations revealed that different mutational mechanisms can lead to the observed in this disease, and this result warrants consideration 1–7 phenotype of cone photoreceptor dysfunction. in the design of future clinical trials that would include both Two frequently encountered types of mutations causing genotypes. BCM are as follows: (1) large deletions covering the locus control region and/or parts of the OPN1LW and OPN1MW or the entire gene cluster, thereby preventing expression METHODS of the OPN1LW/OPN1MW genes; and (2) the C203R missense mutation.1,8,9 We first studied BCM patients with large deletion Human Subjects mutations and have provided evidence that although cone photoreceptor cells are diminished from early life, there are This study was approved by the University of Pennsylvania sufficient numbers remaining across the central retina, albeit Institutional Review Board; informed consent was obtained with shortened outer segments, to suggest the potential value from adults, and assent with parental permission for all of gene augmentation .10 Our next studies inquired children. The study procedures adhered to the tenets of the about possible outcome measures for future clinical trials of Declaration of Helsinki. The cohort included 34 patients therapy and again mainly involve BCM due to deletion (age range, 5–70 years) from 20 families (Table), all of mutations.11,12 whom had a clinical diagnosis of BCM and either a large

Copyright 2018 The Authors iovs.arvojournals.org j ISSN: 1552-5783 5762

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded from iovs.arvojournals.org on 09/27/2021 BCM Genotypes IOVS j December 2018 j Vol. 59 j No. 15 j 5763

TABLE. Clinical Characteristics of BCM Patients With Deletion and Missense Mutations

Age at Visual Patient/Family in Patient/Family Visits, y Acuity* Refraction† Mutation Class: Mutation Detail Previous Publications‡

Deletion mutations P1/F1 5, 7 20/200 2.50 Deletion: LCR, OPN1LW (partial) P7/F3 P2/F2 5, 8 20/100 13.00 Deletion: OPN1LW, OPN1MW (partial) P1/F1 P3/F2 8, 10 20/80 5.00 Deletion: OPN1LW, OPN1MW (partial) P2/F1 P4/F3 7, 12 20/100 6.75 Deletion: LCR, OPN1LW P5/F2 P5/F2 11, 13 20/63 9.25 Deletion: OPN1LW, OPN1MW (partial) P3/F1 P6/F4 13 20/100 6.00 Deletion: LCR, OPN1LW (partial) n/a P7/F3 12, 16 20/80 7.25 Deletion: LCR, OPN1LW P6/F2 P8/F5 14, 17 20/100 4.75 Deletion: LCR, OPN1LW P15/F6 P9/F6 18 20/125 11.50 Deletion: LCR, OPN1LW P25/F15 P10/F1 10, 19 20/100 6.25 Deletion: LCR, OPN1LW (partial) P8/F3 P11/F6 24 20/80 8.75 Deletion: LCR, OPN1LW P26/F15 P12/F1 16, 25 20/80 9.00 Deletion: LCR, OPN1LW (partial) P9/F3 P13/F1 19, 28, 31 20/100 7.00 Deletion: LCR, OPN1LW (partial) P10/F3 P14/F7 30 20/250 10.25 Deletion: LCR, OPN1LW n/a P15/F8 28, 32 20/125 7.25§ Deletion: LCR, OPN1LW P16/F7 P16/F9 33 20/100 3.25§ Deletion: LCR, OPN1LW (partial) P17/F8 P17/F10 35, 40 20/80 6.50 Deletion: LCR, OPN1LW P18/F9 P18/F11 32, 43, 50 20/100 6.25 Deletion: LCR, OPN1LW P19/F10 P19/F2 48, 50 20/125 11.00jj Deletion: OPN1LW, OPN1MW (partial) P4/F1 P20/F12 55 20/63 6.75 Deletion: LCR, OPN1LW P20/F11 P21/F13 60 20/160 5.00jj Deletion: LCR, OPN1LW, OPN1MW n/a Missense C203R mutation P22/F14 5 20/125 9.75 Missense: C203R n/a P23/F15 14 20/80 4.50 Missense: C203R n/a P24/F16 13, 16 20/80 6.50 Missense: C203R P28/F17 P25/F15 18 20/80 2.25 Missense: C203R n/a P26/F17 24 20/100 5.50 Missense: C203R n/a P27/F18 34 20/100 11.00 Missense: C203R# n/a P28/F18 36 20/100 6.00 Missense: C203R n/a P29/F16 35, 38 20/80 6.25 Missense: C203R P29/F17 P30/F19 39, 42 20/100 1.00 Missense: C203R P27/F16 P31/F15 52 20/80 6.50 Missense: C203R n/a P32/F18 53 20/100 1.50 Missense: C203R n/a P33/F14 59 20/160 9.75¶ Missense: C203R# n/a P34/F20 70 20/100 9.25 Missense: C203R n/a LCR, locus control region; OPN1LW, long-wave–sensitive opsin-1 gene; OPN1MW, medium-wave–sensitive opsin-1 gene; n/a, not applicable. * Best-corrected visual acuity in the test eye for OCT at most recent visit. † Refractive status of the test eye for OCT at most recent visit, given as spherical equivalent. ‡ Cideciyan et al.,10 (2013); Luo et al.,11 (2015). § Staphyloma limited to optic disc region. jj Foveal atrophic changes. ¶ Chorioretinal atrophic patches outside macula. # Genetic analysis was not performed for these patients, but a family member has the given mutation.

deletion mutation in the OPN1LW/OPN1MW gene array Optical Coherence Tomography (n ¼ 21, ages 5–60 years) or the C203R missense mutation (n ¼ 13, ages 5–70 years). Longitudinal data were available OCT imaging was performed in one eye from each of the 34 for 14 patients with the deletion mutation (mean follow-up BCM patients. Cross-sectional images of the retina were duration, 5.7 years; median follow-up duration, 4 years; captured by using mainly spectral-domain OCT (SD-OCT) range of follow-up duration, 2–18 years spanning ages 5–50 devices (RTvue-100; Optovue, Inc., Fremont, CA, USA) and, in years) and for three patients with the C203R mutation a few instances, time-domain OCT (TD-OCT) instruments (patients had 3-year follow-up durations spanning ages 13– (OCT1, OCT3; Carl Zeiss Meditec, Dublin, CA, USA) as 10 42 years). All subjects underwent a complete ophthalmic described. Briefly, overlapping 4.5- or 9-mm scans were exam including best-corrected visual acuity measured with taken along the horizontal and vertical meridians through the the Early Treatment Diabetic Retinopathy Study (ETDRS) fovea. Segmentation analysis was performed with custom methodology. ETDRS acuity of the eye imaged with optical computer programs (MatLab 7.5; MathWorks, Inc., Natick, coherence tomography (OCT) from the most recent visit MA, USA), and signal peaks corresponding to retinal laminae was compared between the two cohorts of patients with were assigned on the basis of previously published work.13–16 different genotypes: the C203R mutation (n ¼ 13) and The foveola was identified as the maximum depression on the deletion mutation (n ¼ 21). was not scans, and foveal outer nuclear layer (ONL) thickness was performedaspartofthisstudy. analyzed as in previous works.10 Scans from normal subjects

Downloaded from iovs.arvojournals.org on 09/27/2021 BCM Genotypes IOVS j December 2018 j Vol. 59 j No. 15 j 5764

(n ¼ 22; age range, 8–62 years) for comparison with those of At each fixed background illuminance, a series of stimulus patients have been previously published and include a normal gratings of decremental retinal illuminance was presented. For subset (n ¼ 15) with spherical errors of þ2to4 diopters (D), each background and stimulus illuminance combination, the and a subset (n ¼ 7) with higher degrees of myopia (5to10 subjects were shown gratings of a range of spatial frequencies D).10 ONL and rod outer segment (ROS) thicknesses were (0.3–6.4 cyc/deg, 0.5-second duration) oriented at either 458 or measured at the rod hot spot, a rod-rich region 108 to 168 (3–5 1358, and were asked to choose a direction (i.e., two- mm) eccentric to the fovea in the superior retina.17 Each was alternative forced-choice paradigm). Ten trials were shown at defined as the average of five measurements taken at 0.58 each stimulus spatial frequency and the threshold was defined intervals over a region 10.58 to 12.58 eccentric to the fovea in as the highest spatial frequency with at least 90% correct the superior retina.18 ONL thickness was plotted at the fovea, responses. If the subject discriminated the finest spatial and a generalized additive model was used to describe frequency available (6.4 cyc/deg), the threshold was consid- photoreceptor loss over time.19 To take into account multiple ered indeterminate. If the subject could not discriminate the recordings from the same subject, random effect was coarsest spatial frequency (0.3 cyc/deg), the stimulus was incorporated in the model, and rate of decay was calculated presented without a grating (zero spatial frequency; a disc of from this model. Statistical analysis was performed by using R peak intensity) in order to determine the detectability of the statistical computing software (version 3.4.4; Vienna, Austria). increment under those conditions. The extent of the disruption of the hyperreflective layer Grating acuities in central and nasal retinas were tested in demarcating the inner segment/outer segment (IS/OS) line was each subject with appropriate fixation. Grating stimuli larger analyzed; horizontal raster scans of the macula (6 3 6 mm, 101 than the typical extent of nystagmus11 were used. Specifically, B-scans, 513 A-scans) were obtained with SD-OCT; and several in central testing, fixation was to the center of four dots placed single scans going through the fovea (4.5 mm) with TD-OCT. at the parafovea at 3-, 6-, 9-, and 12-o’clock positions, and a Extent of disruption of the IS/OS line was measured as the stimulus grating of 78 in diameter was presented. For nasal distance between edges of intact IS/OS. In cases of multiple retinal testing, fixation was to a single dot placed 228 nasal to disruptions over the area, the longest extent was taken. To be the fovea and a stimulus grating of 118 in diameter was shown. noted, axial length estimates were not available; however, all The background was uniform across 328 in diameter in both lateral distances are presented in terms of visual angle, which is central and nasal testing. expected to be independent from ocular magnification differences between patients. RESULTS Static Automated Perimetry Structural Assessments A modified computerized perimeter (Humphrey Field Analyzer [HFA], HFA-750i analyzer; Zeiss-Humphrey, Dublin, CA, An OCT image from a representative 22-year-old normal USA)20,21 was used to measure retinal sensitivity across the subject (Fig. 1A) and OCT scans across the vertical meridian vertical meridian in patients with the C203R mutation (n ¼ 12) through the fovea are shown for BCM patients, representing and with a deletion mutation (n ¼ 7). Photopic conditions the C203R mutation (Fig. 1B) and the large deletion (Fig. 1C) tested were an achromatic white stimulus on a 10 cd/m2 white cohorts, selected for comparable ages. A qualitative compar- background and a 440-nm stimulus on a 100 cd/m2 yellow ison of the scans from the two groups showed similarity at background. Scotopic testing was also conducted in dark- early ages. The two 5-year-olds, P22 and P1, as well as the 13- adapted eyes by using a 500-nm stimulus. In all tests, the year-olds, P24 and P5, had ONL of comparable thickness and stimulus duration was 200 ms; the stimulus size was Goldmann little or no interruption of the IS/OS line. Notable even at these V (1.78 diameter); and pupils were dilated. early ages was the lack of a foveal bulge—indicating a decrease in foveal cone outer segment layer thickness.18,22 At age 24 years, P11 with a large deletion mutation (Fig. 1C) showed Chromatic Grating Acuity some disruption of the IS/OS line and thinning of the foveal Chromatic grating acuities were estimated by using a modified ONL, neither of which were present in P26, the C203R MP1 as previously described12 in patients with the C203R mutation counterpart (Fig. 1B). P17 (age 40 years) with a mutation (n ¼ 12; median age, 31 years; range, 12–60 years) deletion mutation (Fig. 1C) showed disruption of the IS/OS line and with a deletion mutation (n ¼ 9; median age, 37 years; and thinning of the ONL in the central retina; these features range, 14–70 years). In brief, an external microprojector (DLP appear to be absent from P30 (age 39 years) with the C203R Lightcrafter with DLP3000 micromirror array; Texas Instru- mutation (Fig. 1B). At age 52 years, P31 from the C203R ments, Dallas, TX, USA) inserted into the Nidek MP1 scotopic mutation cohort (Fig. 1B) showed disruption of the IS/OS line microperimeter (Nidek Technologies, Padova, Italy) was at the fovea, but foveal ONL thinning was not observed, controlled with an external computer. Gratings of desired whereas P18 (age 50 years) from the large deletion cohort (Fig. color (via the combined output of three light-emitting diodes: 1C) showed pronounced disruption of the IS/OS line and red ¼ 624 nm, green ¼ 526 nm, blue ¼ 454 nm) and retinal substantially decreased foveal ONL thickness. The extent of the illuminance were presented with or without achromatic or IS/OS disruption in P33 (age 59 years) was wider than that of chromatic backgrounds. Luminance intrusion could not be 52-year-old patient P31, both from the C203R mutation group ruled out for high-contrast chromatic gratings presented under (Fig. 1B). Foveal ONL thickness was within normal limits in high photopic conditions. P33, and this was in contrast with the foveal atrophy in P21 at Stimulus and background combinations tested in the age 60 years, representing the large deletion mutation cohort current study were photopic white-on-white (WonW), phot- (Fig. 1C). opic blue-on-yellow (BonY), photopic red-on-blue (RonB), To quantify and compare the outer retinal structure in these scotopic WonW, and scotopic BonY. Photopic conditions cohorts, we measured the ONL thickness in two locations: the referred to a retinal illuminance of 2.2 log phot-Td for the cone-rich foveola, and the rod hot spot. ROS thickness was also white or blue backgrounds, and 2.6 log S-Td for the yellow measured at the rod hot spot. ONL thickness measured at the background. In scotopic testing, the retinal illuminance was 2 foveola for each BCM patient from their available visits was log scot-Td. plotted against age and compared with data from normal

Downloaded from iovs.arvojournals.org on 09/27/2021 BCM Genotypes IOVS j December 2018 j Vol. 59 j No. 15 j 5765

FIGURE 1. Comparison of OCT scans along the vertical meridian through the fovea of BCM patients with the two different genotypes. (A) OCT from a representative normal subject with retinal features labeled: outer plexiform layer (OPL); external limiting membrane (ELM); signal originating near junction between inner and outer segments (IS/OS); signal originating near rod outer segment tips and RPE apical processes (ROST); foveal bulge (FB). Photoreceptor layers are colored for visibility: ONL (dark blue), ROS (light blue). (B) OCT images from representative patients with the C203R mutation. (C) OCT images from representative patients with a large deletion mutation. (B, C) Scans of comparably aged patients between genotype cohorts are ordered from youngest (top) to oldest (bottom). (D) ONL thickness at the foveola is quantified as a function of age. Gray dashed lines

Downloaded from iovs.arvojournals.org on 09/27/2021 BCM Genotypes IOVS j December 2018 j Vol. 59 j No. 15 j 5766

represent the range of normal ONL thickness. For patients with longitudinal data, symbols from each visit are connected with black lines. Thick blue and green lines model ONL decay in the C203R and the large deletion cohorts, respectively; shaded areas represent the 95% confidence interval. (E) ONL thickness at the RHS (rod hot spot) is quantified and plotted against age. Gray dotted lines represent the normal limits for thickness; both genotypes show normal ONL thickness at the RHS irrespective of age. (F) ROS thickness at the RHS is plotted against age for both mutation cohorts; normal limits are shown as gray dotted lines. Both cohorts show decreased thickness relative to normal, with no difference between cohorts. Key (at right in [F]) indicates data from normal subjects: gray circles; from patients with C203R mutation: blue triangles; and from patients with deletion mutations: green squares.

subjects (Fig. 1D). For subjects with serial data, median follow- Changes to the IS/OS line, as noted in these cross-sectional up duration for each cohort was 3 years. Foveal ONL thickness data, were detectable in our limited serial data (Figs. 2D, 2E). in BCM patients with the C203R mutation was either normal P24, a patient with a C203R mutation, showed thinning at age (within 62 SD from the mean of the normal subjects) or 13 years, and by age 16 years, there were multiple further small slightly below normal limits for all patients except for the 70- loci of disruption to the IS/OS line (Fig. 2D). P12, a patient year-old patient, who showed severe thinning. This is in with a large deletion mutation, showed gross disruption of the contrast with the BCM patients with a large deletion mutation, IS/OS line occurring in the interval from age 16 to 25 years most of whom had abnormally thin ONL. A generalized (Fig. 2E). Do rates of the expansion of disruptions in the IS/OS additive model with random effects was used to describe the line differ between the mutation cohorts? The extent of IS/OS change in foveal ONL thickness with time (Fig. 1D, solid lines). line disruptions were quantified and plotted as a function of Before approximately 40 years of age, the rate of change of age (Fig. 2F). A linear mixed-effect model was used to model ONL thickness in the C203R mutation cohort is slightly the growth rate of IS/OS disruption for each group. Despite positive, consistent with what has been observed in a normal similarities between the groups for the first 2 decades of life, population.23,24 Between the fifth and sixth decades of life, extent of IS/OS line disruption for patients with the C203R mutation was estimated to grow at 0.22 per year, whereas for however, the ONL falls below the normal range and eventually 8 patients with a large deletion mutation, growth was signifi- further declines. In contrast, even early in life, ONL thickness cantly faster at 0.788 per year (P < 0.001) (Fig. 2F, upper for BCM due to large deletion mutations is below normal, and panel). In addition, IS/OS defect extent was plotted against the declines at a constant rate of approximately 1 lm/y. percentage of ONL thickness loss (relative to mean, age- We then asked whether there was a difference in foveal matched normal patients). In BCM patients of both cohorts, ONL thickness between cohorts even in young patients, that is, when ONL thickness was normal (defined as 62 SD of normal those <20 years of age (10 patients with large deletion mean), the extent of the IS/OS disruption did not exceed 0.58, mutations and 4 with the C203R mutation). The ONL thickness with the exception of two patients. A patient with a large of those with the C203R mutation (median, 72.3 lm; range, deletion mutation, P13, at 28 and 31 years, showed an extent 71–89 lm) and those with a large deletion mutation (median, of IS/OS line disruption of 1.58 and 1.758, respectively; and 50.7 lm; range, 41–66 lm) were significantly different (P ¼ P33, a 59-year-old with the C203R mutation, had an IS/OS line 0.006, Mann-Whitney test). At the rod hot spot, there were no disruption extent of 1.88. Piecewise linear modeling (Fig. 2F, significant differences in rod ONL thickness or ROS layer lower panel, gray dashed lines) illustrates that the size of the thickness between the C203R and the large deletion cohorts, IS/OS line disruption is generally stable until ONL thickness although most BCM patients showed abnormally reduced ROS loss exceeds 56%; extent then increases at a significantly faster thickness, which did not vary significantly with age (Figs. 1E, rate of 0.998 per 10% loss of ONL thickness (P < 0.001). 1F). These results are consistent with our previous reports OCT scans representing a proposed sequence of five stages quantifying ROS layer thickness of BCM patients with deletion of disease progression in the central retina of BCM are shown mutations.10 The average ROS thickness at the rod hot spot for (Fig. 3A); schematics of the stages are also depicted (Fig. 3B). BCM patients, 26.7 lm, was significantly less than the average At stage 1, the BCM foveolar ONL has normal thickness but for normal subjects, 33.7 lm(P < 0.001). For patients with there is thinning of the OS lamina, hence the lack of a foveal multiple visits, values were averaged. bulge. The IS/OS line is intact. At stage 2, the ONL thickness Thesequenceofchangesleadingtolossofcentral remains within normal limits but there is thinning and patchy photoreceptors in BCM patients is shown (Fig. 2). A magnified disruption of the IS/OS line. At stage 3, the foveolar ONL has OCT image centered at the fovea of a 23-year-old normal become borderline reduced in thickness, and the IS/OS line is subject (Fig. 2A) was compared with those of patients with the thinned and becomes more disrupted. At stage 4, foveolar ONL C203R mutation (Fig. 2B) and the large deletion mutation (Fig. is abnormally thin; there are major disruptions to the IS/OS line 2C). There are similarities in the overall pattern of progression and prominent gaps leading to the appearance of a hypore- between the BCM cohorts. OCT scans of P25, age 18 years, in flective zone. Stage 5 represents an atrophic central retina. The the C203R cohort (Fig. 2B), and P5, age 13 years, in the large difference in progression of the two genotypes is also shown schematically (Fig. 3C). It is postulated that stages 2 and 3 are deletion cohort (Fig. 2C), both depict a disruption of the IS/OS more prolonged in BCM patients with the C203R mutation. line (yellow lines mark the edges of the broadest disruption of Stage 2 may extend 3 decades, and stage 3 approximately 2 the IS/OS band). In P11 (age 24 years, deletion mutation; Fig. decades. This is in contrast to the single decade for these stages 2C) and P33 (age 59 years, C203R mutation; Fig. 2B), the in BCM patients with a large deletion mutation who would region of disrupted IS/OS line widened, and there was at least have a more rapidly progressive course. Very late in life, the one other locus at which the IS/OS line had thinned (indicated two time courses would be postulated to converge with foveal by yellow arrows). Thinning appears to precede full IS/OS line atrophy as a final common late stage. disruption. Multiple loci of disrupted IS/OS line can lead to large hyporeflective gaps beneath the fovea (P34, age 70 years, C203R mutation, Fig. 2B; and P18, age 50 years, deletion Functional Assessments mutation, Fig. 2C). In the deletion cohort, central retinal We asked whether the OCT segmentation results and the atrophy was observed (P19, age 50 years; Fig. 2C). The C203R observed difference in rate of foveal photoreceptor nuclear cell patients in this study were not observed to progress to this loss were also detectable as a difference in visual acuity in the stage. two cohorts of patients. The best-corrected visual acuity for

Downloaded from iovs.arvojournals.org on 09/27/2021 BCM Genotypes IOVS j December 2018 j Vol. 59 j No. 15 j 5767

FIGURE 2. Central OCT scans along the horizontal meridian through the fovea in the two genotypes illustrating defects in IS/OS line. (A) OCT of a representative normal subject. (B) Sequence of OCT images showing the general pattern of progression of IS/OS line disruption in patients with the C203R mutation. (C) Sequence of OCT images illustrating pattern of progression of IS/OS line disruption in patients with large deletions. Points of thinning are marked with yellow arrows; yellow lines delimit the extents of disrupted areas. Green squares, patients with large deletion mutations; blue triangles, patients with C203R mutations. (D) Serial OCT scans from a C203R BCM patient (P24) show progression from one locus of IS/OS thinning to many small regions of disruption over a 3-year interval. (E) Serial scans from a BCM patient (P12) with a large deletion mutation show

Downloaded from iovs.arvojournals.org on 09/27/2021 BCM Genotypes IOVS j December 2018 j Vol. 59 j No. 15 j 5768

the increase in extent and severity of the disrupted IS/OS band over an interval of 9 years. (F) Upper panel: Extent of the IS/OS line defect is plotted against age; for patients with multiple visits, visits are connected by a solid black line. Blue (C203R mutation patients) and green (patients with large deletion mutations) dotted lines model defect extent growth over time for each mutation cohort. Lower panel: Extent of the IS/OS line defect size is also plotted against the percentage of ONL loss relative to normal, age-matched subjects. Piecewise linear model (gray dashed lines) shows that for both cohorts, IS/OS defect size remains small and relatively stable until 56% of ONL thickness is lost (arrow), after which IS/OS defect size increases with further ONL thinning.

the latest visit of each patient (and corresponding to the age significantly different from zero (P ¼ 0.53, multivariate and eye of the OCT results) was studied. There was no regression). A comparison of refractive errors in the deletion significant difference in acuities between the two cohorts (P ¼ versus C203R mutation cohorts in our population (Table) 0.15, Mann-Whitney test), and they were found to be similarly showed that the average degree of myopia in the deletion distributed (P ¼ 0.2, Kolmogorov-Smirnov test). Ages were cohort (7.30 6 2.67 D) was slightly greater than that in the comparable for both groups; median ages were 36 and 24 years C203R patient cohort (6.14 6 3.24 D), but the difference for C203R and the large deletion cohorts, respectively. Group between the groups was not statistically significant (t-test, P ¼ mean acuities were 0.68 logMAR for the C203R mutation 0.25). cohort and 0.71 logMAR for the large deletion cohort; the To better understand the photoreceptors contributing to difference was not significant (P ¼ 0.56, controlling for age). the visual acuity measures, we performed specialized tests in a Acuities were plotted as a function of age and compared to subset of patients with the C203R mutation (n ¼ 12) and large normal25 (Fig. 4A). The rates of change of visual acuity with age deletions (n ¼ 9). First we evaluated perimetric light sensitivity were 0.0012 and 0.00057 logMAR/y, respectively, and not along the vertical meridian. As expected from previous

FIGURE 3. Stages of BCM disease progression. (A) Representative OCT images from BCM patients show each of five proposed stages of progression. Patient numbers are given; scans from patients with C203R mutations, blue triangles; scans from patients with deletion mutations, green squares. Scans are horizontal and centered on the fovea (F). (B) Schematic representations of the changes in retinal architecture that are observed in BCM patients at different stages. Retinal laminae are color coded and labeled (ONL, blue; ELM, yellow; IS, dark gray; IS/OS, red; OS, light gray; COST, brown; RPE, orange; BrM, tan). The normal panel (far left) has an illustration of the retinal cell types. Stages 1 to 5 depict the progression of BCM disease (for the red IS/OS line, white boxes indicate points of thinning or small disruptions; black boxes with red dashes indicate hyporeflectivity zones (cavitation). (C) Models of progression in the C203R and the large deletion mutation cohorts. C203R mutation patients progress more slowly, with stages 2 and 3 being prolonged as compared to the large deletion mutation patient pattern. BrM, Bruch’s membrane; COST, cone outer segment tips; N, nasal retina; RPE, retinal pigment epithelium; T, temporal retina.

Downloaded from iovs.arvojournals.org on 09/27/2021 BCM Genotypes IOVS j December 2018 j Vol. 59 j No. 15 j 5769

FIGURE 4. Visual function in BCM due to C203R mutation (blue triangles) or large deletion mutations (green squares). (A) Visual acuity expressed in logMAR as a function of age. Linear regressions and 95% confidence intervals are shown as solid and dashed lines, respectively; normal values derived from Elliott et al.25 (1995). (B) Retinal sensitivity across the vertical meridian for photopic white-on-white (WonW, left), photopic blue-on- yellow (BonY, middle), and scotopic blue (B, right) stimuli. (C, D) Spatial frequencies of gratings resolved in the central retina for different increments over a range of photopic (WonW, and red-on-blue [RonB]), S-cone (BonY), or scotopic (WonW) backgrounds. Results from normal eyes that could resolve the finest grating (6.4 cyc/deg) available for the current instrument are designated with ^. Results from BCM eyes that could not resolve the coarsest grating (0.3 cyc/deg) available are designated with 3 (symbols are jittered for better visualization). Inset: Schematic of a left eye showing the optic nerve and major blood vessels. Fixation is to the middle of the four black squares and the gray circle with black outline shows the central localization of the gratings presented for 0.5 seconds. (E, F) Spatial frequencies of gratings resolved in the peripheral retina. Other details as per (C) and (D). Inset: Schematic of a left eye with fixation (single black square) and stimulus (gray circle with black outline) showing the nasal retinal localization of the gratings presented. All error bars are 6 SEM. Gray dashed lines depict 95% CI of normal.

Downloaded from iovs.arvojournals.org on 09/27/2021 BCM Genotypes IOVS j December 2018 j Vol. 59 j No. 15 j 5770

studies,11 sensitivity to white stimuli presented on a white Among the more common genotypes reported are large background was abnormally reduced by an average of 12.2 dB deletions and the C203R missense mutation. Concern has in C203R mutation and by 11.2 dB in large deletion mutation been raised that the mechanism of disease resulting from the patients across retinal locations; there was no significant missense C203R mutation could involve misfolded cone difference between the cohorts (P ¼ 0.22; Fig. 4B, left). with potential for a toxic effect on the photoreceptors, Sensitivity to 440-nm stimuli presented on a yellow back- comparable to some missense mutations in .33,34 It ground straddled the lower limits of normal (mean losses of 4.4 is of interest that two individuals with red/green color and 4.7 dB for the C203R and the large deletion cohorts, deficiency and molecular evidence of no OPN1LW genes but respectively) and suggested near-normal S-cone function; there a C203R mutation encoded by one of the OPN1MW genes in was no significant difference between the cohorts (P ¼ 0.82; the photopigment arrays had disruption of the cone photore- Fig. 4B, middle). Similarly, rod function measured with a 500- ceptor mosaic, and this was postulated to cause early nm stimulus under dark-adapted conditions was normal and degeneration of some of the cone cells but with retained there was no significant difference between the two genotypes normal acuity.33 (P ¼ 0.89; Fig. 4B, right). There is no consensus in the literature about the level of A previously developed modified MP1 was used to evaluate severity of the BCM phenotype associated with the C203R the spatial resolution in BCM retinas over scotopic and genotype.6,11,27,30,31 Based on the hypothesis of a possible photopic conditions with achromatic or chromatic gratings.12 toxic effect of the missense mutation and a more aggressive In the central retina, using achromatic low-contrast gratings on cone disease in such patients,6 we studied patients with the an achromatic photopic background (WonW), the finest C203R genotype and compared the results with a cohort of gratings resolved by the BCM patients were ~3.0 cyc/deg patients of similar age range but with large deletion mutations. (corresponding to 20/200), which was abnormal; there was no We previously have demonstrated that in human BCM with difference between genotypes (P ¼ 0.52) (Fig. 4C, left). Using deletion mutations there are sufficient residual foveal L/M- red gratings on a blue photopic background, BCM patients cones to warrant a approach.10 In addition, there could not detect the orientation for any available level of has been recent proof-of-concept of gene augmentation contrast, implying a severe loss of L/M-cone–mediated input therapy delivered subretinally in the Opn/mw/ mouse 35 (Fig. 4C, middle). Using blue gratings on a yellow photopic mutant. Thestepwetooktowardunderstandingthe background, BCM patients’ results were normal (Fig. 4C, right) common C203R missense mutation was to ask if the human and showed similar spatial frequency to WonW conditions. phenotype differed dramatically from that of patients with Under the scotopic condition, resolution of WonW gratings deletion mutations. A necessary parallel step would be to was within normal limits in both BCM cohorts (Fig. 4D); there develop and characterize a murine model of the C203R was no difference between the two genotypes across the mutation and then perform proof-of-concept experiments as increments above background tested (P ¼ 0.59). BonY grating was done with the knockout and make that comparison. acuity was also tested under scotopic conditions and showed The unexpected result in the present work was that disease similar results to scotopic WonW (data not shown). The results progression in patients with the C203R genotype appeared less taken together support the hypothesis that S-cones were likely aggressive than that in our cohort of patients with deletion resolving the gratings in the central retina under high-photopic mutations. There was a definite difference in the foveal cone low-contrast conditions in both genotypes of BCM patients. layer thickness across the age spectrum Grating acuities were also tested at the nasal retina where L/ studied in cross-sectional data bolstered by limited longitudinal M-cone contribution in normal retinas would be expected to data. The prolonged period of foveal ONL retention extended be lower. With targets at low increments from background, over approximately 3 decades of life (from approximately ages normal resolution to WonW and RonB stimuli were within the 20–50 years) in the patient cohort with the C203R mutation measurement range of the instrument designed for low vision. versus the cohort with large deletion mutations. Older patients Both genotypes of BCM patients showed abnormally reduced of both genotypes converged to the same advanced stage of photopic WonW results. Under RonB conditions, BCM patients foveal cone loss in their seventh decade of life. This may could not resolve the gratings, whereas under BonY condi- represent a difference in rates of progression of the two tions, the results were within normal limits. Furthermore, genotype groups or, alternatively, a later onset but similar rate comparable to the central retina, scotopic WonW and BonY of progression in C203R versus deletion mutation groups. grating acuities were within normal limits (Fig. 4F, only WonW It could be asked why a simple and traditional outcome of shown). There was no difference in peripheral grating acuity vision, such as visual acuity, did not show a comparable between the two genotypes in all the conditions tested. The difference in progression rates between genotypes. At the results taken together support the hypothesis that S-cones earliest ages studied in both cohorts of patients, there is a lack were likely resolving the gratings in the nasal retina under high of a foveal bulge, a clinical marker for reduced foveal cone OS photopic conditions in both genotypes of BCM patients. length. The lack of a foveal bulge is associated with reduced visual acuity in reports of surgical recovery after macula-off retinal detachment.22,36 The photoreceptor outer segment DISCUSSION disruption becomes more complex with age but it is likely that the cone OS abnormality from early life is at least one reason The molecular complexity of the L- and M-cone opsin genes for reduced acuity, no matter how much further damage is (OPN1LW and OPN1MW) has been explored not only to incurred from the advancing disease process. Further, in understand the basis of but also to elucidate the keeping with the reduced acuity throughout the disease molecular genetic causes of X-linked cone diseas- course, we have previously used chromatic stimuli and fundus es.1,3,6,9,10,11,26–29 BCM causes decreased L/M-cone vision due perimetry to position the target on the anatomical fovea in to simultaneous OPN1LW and OPN1MW gene defects as a BCM patients mainly with the deletion genotype. The results result of one of multiple mutation mechanisms, such as large indicate that almost all of these patients rely on S-cones and deletions involving upstream regulatory sequences (i.e., locus rods for perception11 with rare exception.10 It would therefore control region and promoters) and/or parts of or the entire not be expected to have the progression rate of foveal L/M- OPN1LW and OPN1MW genes, missense mutations or non- cone structure abnormalities be reflected in the function of sense mutations, and L/M interchange mutations.7,9,26,30–32 these other receptor types. In the current study, a comparison

Downloaded from iovs.arvojournals.org on 09/27/2021 BCM Genotypes IOVS j December 2018 j Vol. 59 j No. 15 j 5771

was made of the spatial resolving ability between the two 12. Cideciyan AV, Roman AJ, Jacobson SG, et al. Developing an genotypes, using chromatic gratings under fundus viewing. outcome measure with high luminance for optogenetics There was no difference in results between the two genotypes. treatment of severe retinal degenerations and for gene What are the clinical trial implications of this observed therapy of cone diseases. Invest Ophthalmol Vis Sci. 2016; difference in rate of foveal structural changes between 57:3211–3221. genotypes? The inclusion of BCM patients with the C203R 13. Huang Y, Cideciyan AV, Papastergiou GI, et al. Relation of missense mutation in a gene augmentation trial still awaits optical coherence tomography to microanatomy in normal proof-of-concept experimental results, such as have been and rd chickens. Invest Ophthalmol Vis Sci. 1998;39:2405– reported for a model of deletion mutations, namely, the 2416. 35 knockout mouse. Also, confirmation of the current human 14. Jacobson SG, Aleman TS, Sumaroka A, et al. Disease findings with serial data of foveal photoreceptor structural boundaries in the retina of patients with Usher syndrome preservation until late in life in BCM with the C203R genotype caused by MYO7A gene mutations. Invest Ophthalmol Vis would be important. A natural history study extending decades Sci. 2009;50:1886–1894. is obviously not practical, but longitudinal OCT measurements 15. Spaide RF, Curcio CA. Anatomical correlates to the bands seen in a cohort of BCM patients in their sixth or seventh decade of in the outer retina by optical coherence tomography: life should be feasible. The present findings suggest that there literature review and model. Retina. 2011;31:1609–1619. is a much wider window of opportunity to treat BCM patients 16. Kocaoglu OP, Lee S, Jonnal RS, et al. Imaging cone photore- with the C203R missense mutation than there may be with the ceptors in three dimensions and in time using ultrahigh large deletion genotype. resolution optical coherence tomography with adaptive optics. Biomed Opt Express. 2011;2:748–763. Acknowledgments 17. Curcio CA, Sloan KR, Kalina RE, et al. Human photoreceptor Supported by BCM Families Foundation. topography. J Comp Neurol. 1990;292:497–523. Disclosure: A. Sumaroka, None; A.V. Garafalo, None; A.V. 18. Jacobson SG, Cideciyan AV, Sumaroka A, et al. Defining Cideciyan, None; J. Charng, None; A.J. Roman, None; W. Choi, outcomes for clinical trials of Leber congenital amaurosis None; S. Saxena, None; V. Aksianiuk, None; S. Kohl, None; B. caused by GUCY2D mutations. Am J Ophthalmol. 2017;177: Wissinger, None; S.G. Jacobson, None 44–57. 19. Hastie T, Tibshirani R. Generalized additive models. Statist Sci. 1986;1:297–318. References 20. Jacobson SG, Voigt WJ, Parel JM, et al. Automated light- and 1. Nathans J, Davenport CM, Maumenee IH, et al. Molecular dark-adapted perimetry for evaluating retinitis pigmentosa. genetics of human blue cone monochromacy. Science. 1989; Ophthalmology. 1986;93:1604–1611. 245:831–838. 21. Roman AJ, Schwartz SB, Aleman TS, et al. Quantifying rod 2. Wang Y, Macke JP, Merbs SL, et al. A locus control region photoreceptor-mediated vision in retinal degenerations: dark- adjacent to the human red and green visual pigment genes. adapted thresholds as outcome measures. Exp. Eye Res. 2005; Neuron. 1992;9:429–440. 80:259–272. 3. Nathans J, Maumenee IH, Zrenner E, Sadowski B, et al. 22. Hasegawa T, Ueda T, Okamoto M, et al. Relationship between Genetic heterogeneity among blue-cone monochromats. Am J presence of foveal bulge in optical coherence tomographic Hum Genet. 1993;53:987–1000. images and visual acuity after rhegmatogenous retinal 4. Kellner U, Wissinger B, Tippmann S, et al. Blue cone detachment repair. Retina. 2014;34:1848–1853. monochromatism: clinical findings in patients with mutations 23. Jacobson SG, Aleman TS, Cideciyan AV, et al. Human cone in the red/green opsin gene cluster. Graefes Arch Clin Exp photoreceptor dependence on RPE65 isomerase. Proc Natl Ophthalmol. 2004;242:729–735. Acad Sci U S A. 2007;104:15123–15128. 5. Gardner JC, Michaelides M, Holder GE, et al. Blue cone 24. Perez-Garc´ ´ıa D, Ibanez-Alperte˜ J, Remon´ L, et al. Study of monochromacy: causative mutations and associated pheno- spectral-domain optical coherence tomography in children: types. Mol Vis. 2009;15:876–884. normal values and influence of age, sex, and refractive status. 6. Gardner JC, Liew G, Quan YH, et al. Three different cone Eur J Ophthalmol. 2018;26:135–141. opsin gene array mutational mechanisms; genotype-pheno- 25. Elliott DB, Yang KC, Whitaker D. Visual acuity changes type correlation and functional investigation of cone opsin throughout adulthood in normal, healthy eyes: seeing beyond variants. Hum Mutat. 2014;35:1354–1362. 6/6. Optom Vis Sci. 1995;72:186–191. 7. Yatsenko SA, Bakos HA, Vitullo K, et al. High-resolution 26. Nathans J, Piantanida TP, Eddy RL, et al. Molecular genetics of microarray analysis unravels complex Xq28 aberrations in inherited variation in human color vision. . 1986;232: patients and carriers affected by X-linked blue cone mono- Science 203–210. chromacy. Clin Genet. 2016;89:82–87. 8. Kazmi MA, Sakmar TP, Ostrer H. Mutation of a conserved 27. Carroll J, Dubra A, Gardner JC, et al. The effect of cone opsin in the X-linked cone opsins causes color vision mutations on retinal structure and the integrity of the deficiencies by disrupting protein folding and stability. Invest photoreceptor mosaic. Invest Ophthalmol Vis Sci. 2012;53: Ophthalmol Vis Sci. 1997;38:1074–1081. 8006–8015. 9. Buena-Atienza E, Ruther¨ K, Baumann B, et al. De novo 28. Aboshiha J, Dubis AM, Carroll J, et al. The cone dysfunction intrachromosomal gene conversion from OPN1MW to syndromes. Br J Ophthalmol. 2016;100:115–121. OPN1LWinthemalegermlineresultsinbluecone 29. Orosz O, Rajta I, Vajas A, et al. Myopia and late-onset monochromacy. Sci Rep. 2016;6:28253. progressive associate to LVAVA/MVAVA exon 3 10. Cideciyan AV, Hufnagel RB, Carroll J, et al. Human cone visual interchange haplotypes of opsin genes on chromosome X. pigment deletions spare sufficient photoreceptors to warrant Invest Ophthalmol Vis Sci. 2017;58:1834–1842. gene therapy. Hum Gene Ther. 2013;24:993–1006. 30. Michaelides M, Johnson S, Simunovic MP, et al. Blue cone 11. Luo X, Cideciyan AV, Iannaccone A, et al. Blue cone monochromatism: a phenotype and genotype assessment monochromacy: visual function and efficacy outcome mea- with evidence of progressive loss of cone function in older sures for clinical trials. PLoS One. 2015;10:e0125700. individuals. Eye. 2005;19:2–10.

Downloaded from iovs.arvojournals.org on 09/27/2021 BCM Genotypes IOVS j December 2018 j Vol. 59 j No. 15 j 5772

31. Mizrahi-Meissonnier L, Merin S, Banin E, et al. Variable retinal 34. Athanasiou D, Aguila M, Bellingham J, et al. The molecular and phenotypes caused by mutations in the X-linked photopig- cellular basis of rhodopsin retinitis pigmentosa reveals ment gene array. Invest Ophthalmol Vis Sci. 2010;51:3884– potential strategies for therapy. Prog Retin Eye Res. 2018; 3892. 62:1–23. 32. Greenwald SH, Kuchenbecker JA, Rowlan JS, et al. Role of a 35. Deng WT, Li J, Zhu P, et al. Human L- and M-opsins restore M- dual splicing and amino acid code in myopia, cone cone function in a mouse model for human blue cone dysfunction and cone dystrophy associated with L/M opsin interchange mutations. Trans Vis Sci Tech. 2017;6(3):2. monochromacy. Mol Vis. 2018;24:17–28. 33. Carroll J, Baraas RC, Wagner-Schuman M, et al. Cone 36. Kobayashi M, Iwase T, Yamamoto K, et al. Association photoreceptor mosaic disruption associated with Cys203Arg between photoreceptor regeneration and visual acuity mutation in the M-cone opsin. Proc Natl Acad Sci U S A. following for rhegmatogenous retinal detachment. 2009;106:20948–20953. Invest Ophthalmol Vis Sci. 2016;57:889–898.

Downloaded from iovs.arvojournals.org on 09/27/2021