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

Abstract

Purpose: To compare the phenotype of blue cone monochromacy (BCM) caused by large deletion mutations with those having the C203R missense mutation.

Methods: BCM patients with large deletion mutations (n = 21; age range, 5–60 years), and with the C203R missense mutation (n = 13; age range, 5–70 years), were studied with optical coherence tomography, visual acuity, and perimetric sensitivity in a retrospective 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 adapting conditions with a modified microperimeter.

Results: Both genotypes had abnormal foveal photoreceptor structure early in life. Patients with the C203R mutation, however, had decades-longer persistence of foveal photoreceptor outer nuclear layer thickness and a slower rate of development of inner segment/outer segment defects than did patients with large deletion mutations. At late ages, both genotypes 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 therapies.

Blue cone monochromacy (BCM) is an X-linked disease that is characterized by impaired red (L, long wavelength) and green (M, middle wavelength) cone photoreceptor function caused by mutations in the OPN1LW/OPN1MW gene cluster on chromosome Xq28. Studies of the molecular basis of BCM have revealed that different mutational mechanisms can lead to the phenotype of cone photoreceptor dysfunction.^1–7 

Two frequently encountered types of mutations causing BCM are as follows: (1) large deletions covering the locus control region and/or parts of the OPN1LW and OPN1MW genes or the entire gene cluster, thereby preventing expression of the OPN1LW/OPN1MW genes; and (2) the C203R missense mutation.^1,8,9 We first studied BCM patients with large deletion mutations and have provided evidence that although cone photoreceptor cells are diminished from early life, there are sufficient numbers remaining across the central retina, albeit with shortened outer segments, to suggest the potential value of gene augmentation therapy.¹⁰ Our next studies inquired about possible outcome measures for future clinical trials of therapy and again mainly involve BCM due to deletion mutations.^11,12 

The goal of the current study was to compare the phenotype of the other common BCM genotype—the C203R missense mutation—with that of the large deletion mutations. The results indicate very different disease progressions in BCM due to the two most common types of mutations observed in this disease, and this result warrants consideration in the design of future clinical trials that would include both genotypes. 

Methods

Human Subjects

This study was approved by the University of Pennsylvania Institutional Review Board; informed consent was obtained from adults, and assent with parental permission for all children. The study procedures adhered to the tenets of the Declaration of Helsinki. The cohort included 34 patients (age range, 5–70 years) from 20 families (Table), all of whom had a clinical diagnosis of BCM and either a large deletion mutation in the OPN1LW/OPN1MW gene array (n = 21, ages 5–60 years) or the C203R missense mutation (n = 13, ages 5–70 years). Longitudinal data were available for 14 patients with the deletion mutation (mean follow-up duration, 5.7 years; median follow-up duration, 4 years; range of follow-up duration, 2–18 years spanning ages 5–50 years) and for three patients with the C203R mutation (patients had 3-year follow-up durations spanning ages 13–42 years). All subjects underwent a complete ophthalmic exam including best-corrected visual acuity measured with the Early Treatment Diabetic Retinopathy Study (ETDRS) methodology. ETDRS acuity of the eye imaged with optical coherence tomography (OCT) from the most recent visit was compared between the two cohorts of patients with different genotypes: the C203R mutation (n = 13) and deletion mutation (n = 21). Electroretinography was not performed as part of this study. 

Table

 

Clinical Characteristics of BCM Patients With Deletion and Missense Mutations

 

Optical Coherence Tomography

OCT imaging was performed in one eye from each of the 34 BCM patients. Cross-sectional images of the retina were captured by using mainly spectral-domain OCT (SD-OCT) devices (RTvue-100; Optovue, Inc., Fremont, CA, USA) and, in a few instances, time-domain OCT (TD-OCT) instruments (OCT1, OCT3; Carl Zeiss Meditec, Dublin, CA, USA) as described.¹⁰ Briefly, overlapping 4.5- or 9-mm scans were taken along the horizontal and vertical meridians through the fovea. Segmentation analysis was performed with custom computer programs (MatLab 7.5; MathWorks, Inc., Natick, MA, USA), and signal peaks corresponding to retinal laminae were assigned on the basis of previously published work.^13–16 The foveola was identified as the maximum depression on the scans, and foveal outer nuclear layer (ONL) thickness was analyzed as in previous works.¹⁰ Scans from normal subjects (n = 22; age range, 8–62 years) for comparison with those of patients have been previously published and include a normal subset (n = 15) with spherical errors of +2 to −4 diopters (D), and a subset (n = 7) with higher degrees of myopia (−5 to −10 D).¹⁰ ONL and rod outer segment (ROS) thicknesses were measured at the rod hot spot, a rod-rich region 10° to 16° (3–5 mm) eccentric to the fovea in the superior retina.¹⁷ Each was defined as the average of five measurements taken at 0.5° intervals over a region 10.5° to 12.5° eccentric to the fovea in the superior retina.¹⁸ ONL thickness was plotted at the fovea, and a generalized additive model was used to describe photoreceptor loss over time.¹⁹ To take into account multiple recordings from the same subject, random effect was incorporated in the model, and rate of decay was calculated from this model. Statistical analysis was performed by using R statistical computing software (version 3.4.4; Vienna, Austria). The extent of the disruption of the hyperreflective layer demarcating the inner segment/outer segment (IS/OS) line was analyzed; horizontal raster scans of the macula (6 × 6 mm, 101 B-scans, 513 A-scans) were obtained with SD-OCT; and several single scans going through the fovea (4.5 mm) with TD-OCT. Extent of disruption of the IS/OS line was measured as the distance between edges of intact IS/OS. In cases of multiple disruptions over the area, the longest extent was taken. To be noted, axial length estimates were not available; however, all lateral distances are presented in terms of visual angle, which is expected to be independent from ocular magnification differences between patients. 

Static Automated Perimetry

A modified computerized perimeter (Humphrey Field Analyzer [HFA], HFA-750i analyzer; Zeiss-Humphrey, Dublin, CA, USA)^20,21 was used to measure retinal sensitivity across the vertical meridian in patients with the C203R mutation (n = 12) and with a deletion mutation (n = 7). Photopic conditions tested were an achromatic white stimulus on a 10 cd/m² white background and a 440-nm stimulus on a 100 cd/m² yellow background. Scotopic testing was also conducted in dark-adapted eyes by using a 500-nm stimulus. In all tests, the stimulus duration was 200 ms; the stimulus size was Goldmann V (1.7° diameter); and pupils were dilated. 

Chromatic Grating Acuity

Chromatic grating acuities were estimated by using a modified MP1 as previously described¹² in patients with the C203R mutation (n = 12; median age, 31 years; range, 12–60 years) and with a deletion mutation (n = 9; median age, 37 years; range, 14–70 years). In brief, an external microprojector (DLP Lightcrafter with DLP3000 micromirror array; Texas Instruments, Dallas, TX, USA) inserted into the Nidek MP1 scotopic microperimeter (Nidek Technologies, Padova, Italy) was controlled with an external computer. Gratings of desired color (via the combined output of three light-emitting diodes: red = 624 nm, green = 526 nm, blue = 454 nm) and retinal illuminance were presented with or without achromatic or chromatic backgrounds. Luminance intrusion could not be ruled out for high-contrast chromatic gratings presented under high photopic conditions. 

Stimulus and background combinations tested in the current study were photopic white-on-white (WonW), photopic blue-on-yellow (BonY), photopic red-on-blue (RonB), scotopic WonW, and scotopic BonY. Photopic conditions referred to a retinal illuminance of 2.2 log phot-Td for the white or blue backgrounds, and 2.6 log S-Td for the yellow background. In scotopic testing, the retinal illuminance was −2 log scot-Td. 

At each fixed background illuminance, a series of stimulus gratings of decremental retinal illuminance was presented. For each background and stimulus illuminance combination, the subjects were shown gratings of a range of spatial frequencies (0.3–6.4 cyc/deg, 0.5-second duration) oriented at either 45° or 135°, and were asked to choose a direction (i.e., two-alternative forced-choice paradigm). Ten trials were shown at each stimulus spatial frequency and the threshold was defined as the highest spatial frequency with at least 90% correct responses. If the subject discriminated the finest spatial frequency available (6.4 cyc/deg), the threshold was considered indeterminate. If the subject could not discriminate the coarsest spatial frequency (0.3 cyc/deg), the stimulus was presented without a grating (zero spatial frequency; a disc of peak intensity) in order to determine the detectability of the increment under those conditions. 

Grating acuities in central and nasal retinas were tested in each subject with appropriate fixation. Grating stimuli larger than the typical extent of nystagmus¹¹ were used. Specifically, in central testing, fixation was to the center of four dots placed at the parafovea at 3-, 6-, 9-, and 12-o'clock positions, and a stimulus grating of 7° in diameter was presented. For nasal retinal testing, fixation was to a single dot placed 22° nasal to the fovea and a stimulus grating of 11° in diameter was shown. The background was uniform across 32° in diameter in both central and nasal testing. 

Results

Structural Assessments

An OCT image from a representative 22-year-old normal subject (Fig. 1A) and OCT scans across the vertical meridian through the fovea are shown for BCM patients, representing the C203R mutation (Fig. 1B) and the large deletion (Fig. 1C) cohorts, selected for comparable ages. A qualitative comparison of the scans from the two groups showed similarity at early ages. The two 5-year-olds, P22 and P1, as well as the 13-year-olds, P24 and P5, had ONL of comparable thickness and little or no interruption of the IS/OS line. Notable even at these 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 some disruption of the IS/OS line and thinning of the foveal ONL, neither of which were present in P26, the C203R mutation counterpart (Fig. 1B). P17 (age 40 years) with a deletion mutation (Fig. 1C) showed disruption of the IS/OS line and thinning of the ONL in the central retina; these features appear to be absent from P30 (age 39 years) with the C203R mutation (Fig. 1B). At age 52 years, P31 from the C203R mutation cohort (Fig. 1B) showed disruption of the IS/OS line at the fovea, but foveal ONL thinning was not observed, whereas P18 (age 50 years) from the large deletion cohort (Fig. 1C) showed pronounced disruption of the IS/OS line and substantially decreased foveal ONL thickness. The extent of the IS/OS disruption in P33 (age 59 years) was wider than that of 52-year-old patient P31, both from the C203R mutation group (Fig. 1B). Foveal ONL thickness was within normal limits in P33, and this was in contrast with the foveal atrophy in P21 at age 60 years, representing the large deletion mutation cohort (Fig. 1C). 

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 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.

To quantify and compare the outer retinal structure in these cohorts, we measured the ONL thickness in two locations: the cone-rich foveola, and the rod hot spot. ROS thickness was also measured at the rod hot spot. ONL thickness measured at the foveola for each BCM patient from their available visits was plotted against age and compared with data from normal subjects (Fig. 1D). For subjects with serial data, median follow-up duration for each cohort was 3 years. Foveal ONL thickness in BCM patients with the C203R mutation was either normal (within ±2 SD from the mean of the normal subjects) or slightly below normal limits for all patients except for the 70-year-old patient, who showed severe thinning. This is in contrast with the BCM patients with a large deletion mutation, most of whom had abnormally thin ONL. A generalized additive model with random effects was used to describe the change in foveal ONL thickness with time (Fig. 1D, solid lines). Before approximately 40 years of age, the rate of change of ONL thickness in the C203R mutation cohort is slightly positive, consistent with what has been observed in a normal population.^23,24 Between the fifth and sixth decades of life, however, the ONL falls below the normal range and eventually further declines. In contrast, even early in life, ONL thickness for BCM due to large deletion mutations is below normal, and declines at a constant rate of approximately 1 μm/y. 

We then asked whether there was a difference in foveal ONL thickness between cohorts even in young patients, that is, those <20 years of age (10 patients with large deletion mutations and 4 with the C203R mutation). The ONL thickness of those with the C203R mutation (median, 72.3 μm; range, 71–89 μm) and those with a large deletion mutation (median, 50.7 μm; range, 41–66 μm) were significantly different (P = 0.006, Mann-Whitney test). At the rod hot spot, there were no significant differences in rod ONL thickness or ROS layer thickness between the C203R and the large deletion cohorts, although most BCM patients showed abnormally reduced ROS thickness, which did not vary significantly with age (Figs. 1E, 1F). These results are consistent with our previous reports quantifying ROS layer thickness of BCM patients with deletion mutations.¹⁰ The average ROS thickness at the rod hot spot for BCM patients, 26.7 μm, was significantly less than the average for normal subjects, 33.7 μm (P < 0.001). For patients with multiple visits, values were averaged. 

The sequence of changes leading to loss of central photoreceptors in BCM patients is shown (Fig. 2). A magnified OCT image centered at the fovea of a 23-year-old normal subject (Fig. 2A) was compared with those of patients with the C203R mutation (Fig. 2B) and the large deletion mutation (Fig. 2C). There are similarities in the overall pattern of progression between the BCM cohorts. OCT scans of P25, age 18 years, in the C203R cohort (Fig. 2B), and P5, age 13 years, in the large deletion cohort (Fig. 2C), both depict a disruption of the IS/OS line (yellow lines mark the edges of the broadest disruption of the IS/OS band). In P11 (age 24 years, deletion mutation; Fig. 2C) and P33 (age 59 years, C203R mutation; Fig. 2B), the region of disrupted IS/OS line widened, and there was at least one other locus at which the IS/OS line had thinned (indicated by yellow arrows). Thinning appears to precede full IS/OS line 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 mutation, Fig. 2C). In the deletion cohort, central retinal atrophy was observed (P19, age 50 years; Fig. 2C). The C203R patients in this study were not observed to progress to this stage. 

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 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.

Changes to the IS/OS line, as noted in these cross-sectional data, were detectable in our limited serial data (Figs. 2D, 2E). P24, a patient with a C203R mutation, showed thinning at age 13 years, and by age 16 years, there were multiple further small loci of disruption to the IS/OS line (Fig. 2D). P12, a patient with a large deletion mutation, showed gross disruption of the IS/OS line occurring in the interval from age 16 to 25 years (Fig. 2E). Do rates of the expansion of disruptions in the IS/OS line differ between the mutation cohorts? The extent of IS/OS line disruptions were quantified and plotted as a function of age (Fig. 2F). A linear mixed-effect model was used to model the growth rate of IS/OS disruption for each group. Despite similarities between the groups for the first 2 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 patients with a large deletion mutation, growth was significantly faster at 0.78° per year (P < 0.001) (Fig. 2F, upper panel). In addition, IS/OS defect extent was plotted against the percentage of ONL thickness loss (relative to mean, age-matched normal patients). In BCM patients of both cohorts, when ONL thickness was normal (defined as ±2 SD of normal mean), the extent of the IS/OS disruption did not exceed 0.5°, with the exception of two patients. A patient with a large deletion mutation, P13, at 28 and 31 years, showed an extent of IS/OS line disruption of 1.5° and 1.75°, respectively; and P33, a 59-year-old with the C203R mutation, had an IS/OS line disruption extent of 1.8°. Piecewise linear modeling (Fig. 2F, lower panel, gray dashed lines) illustrates that the size of the IS/OS line disruption is generally stable until ONL thickness loss exceeds 56%; extent then increases at a significantly faster rate of 0.99° per 10% loss of ONL thickness (P < 0.001). 

OCT scans representing a proposed sequence of five stages of disease progression in the central retina of BCM are shown (Fig. 3A); schematics of the stages are also depicted (Fig. 3B). At stage 1, the BCM foveolar ONL has normal thickness but there is thinning of the OS lamina, hence the lack of a foveal bulge. The IS/OS line is intact. At stage 2, the ONL thickness remains within normal limits but there is thinning and patchy disruption of the IS/OS line. At stage 3, the foveolar ONL has become borderline reduced in thickness, and the IS/OS line is thinned and becomes more disrupted. At stage 4, foveolar ONL is abnormally thin; there are major disruptions to the IS/OS line and prominent gaps leading to the appearance of a hyporeflective zone. Stage 5 represents an atrophic central retina. The difference in progression of the two genotypes is also shown schematically (Fig. 3C). It is postulated that stages 2 and 3 are more prolonged in BCM patients with the C203R mutation. Stage 2 may extend 3 decades, and stage 3 approximately 2 decades. This is in contrast to the single decade for these stages in BCM patients with a large deletion mutation who would have a more rapidly progressive course. Very late in life, the two time courses would be postulated to converge with foveal atrophy as a final common late stage. 

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.

Functional Assessments

We asked whether the OCT segmentation results and the observed difference in rate of foveal photoreceptor nuclear cell loss were also detectable as a difference in visual acuity in the two cohorts of patients. The best-corrected visual acuity for the latest visit of each patient (and corresponding to the age and eye of the OCT results) was studied. There was no significant difference in acuities between the two cohorts (P = 0.15, Mann-Whitney test), and they were found to be similarly distributed (P = 0.2, Kolmogorov-Smirnov test). Ages were comparable for both groups; median ages were 36 and 24 years for C203R and the large deletion cohorts, respectively. Group mean acuities were 0.68 logMAR for the C203R mutation cohort and 0.71 logMAR for the large deletion cohort; the difference was not significant (P = 0.56, controlling for age). Acuities were plotted as a function of age and compared to normal²⁵ (Fig. 4A). The rates of change of visual acuity with age were 0.0012 and 0.00057 logMAR/y, respectively, and not significantly different from zero (P = 0.53, multivariate regression). A comparison of refractive errors in the deletion versus C203R mutation cohorts in our population (Table) showed that the average degree of myopia in the deletion cohort (−7.30 ± 2.67 D) was slightly greater than that in the C203R patient cohort (−6.14 ± 3.24 D), but the difference between the groups was not statistically significant (t-test, P = 0.25). 

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 × (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 ± SEM. Gray dashed lines depict 95% CI of normal.

To better understand the photoreceptors contributing to the visual acuity measures, we performed specialized tests in a subset of patients with the C203R mutation (n = 12) and large deletions (n = 9). First we evaluated perimetric light sensitivity along the vertical meridian. As expected from previous studies,¹¹ sensitivity to white stimuli presented on a white background was abnormally reduced by an average of 12.2 dB in C203R mutation and by 11.2 dB in large deletion mutation patients across retinal locations; there was no significant difference between the cohorts (P = 0.22; Fig. 4B, left). Sensitivity to 440-nm stimuli presented on a yellow background straddled the lower limits of normal (mean losses of 4.4 and 4.7 dB for the C203R and the large deletion cohorts, respectively) and suggested near-normal S-cone function; there was no significant difference between the cohorts (P = 0.82; Fig. 4B, middle). Similarly, rod function measured with a 500-nm stimulus under dark-adapted conditions was normal and there was no significant difference between the two genotypes (P = 0.89; Fig. 4B, right). 

A previously developed modified MP1 was used to evaluate the spatial resolution in BCM retinas over scotopic and photopic conditions with achromatic or chromatic gratings.¹² In the central retina, using achromatic low-contrast gratings on an achromatic photopic background (WonW), the finest gratings resolved by the BCM patients were ∼3.0 cyc/deg (corresponding to 20/200), which was abnormal; there was no difference between genotypes (P = 0.52) (Fig. 4C, left). Using red gratings on a blue photopic background, BCM patients could not detect the orientation for any available level of contrast, implying a severe loss of L/M-cone–mediated input (Fig. 4C, middle). Using blue gratings on a yellow photopic background, BCM patients' results were normal (Fig. 4C, right) and showed similar spatial frequency to WonW conditions. Under the scotopic condition, resolution of WonW gratings was within normal limits in both BCM cohorts (Fig. 4D); there was no difference between the two genotypes across the increments above background tested (P = 0.59). BonY grating acuity was also tested under scotopic conditions and showed similar results to scotopic WonW (data not shown). The results taken together support the hypothesis that S-cones were likely resolving the gratings in the central retina under high-photopic low-contrast conditions in both genotypes of BCM patients. 

Grating acuities were also tested at the nasal retina where L/M-cone contribution in normal retinas would be expected to be lower. With targets at low increments from background, normal resolution to WonW and RonB stimuli were within the measurement range of the instrument designed for low vision. Both genotypes of BCM patients showed abnormally reduced photopic WonW results. Under RonB conditions, BCM patients could not resolve the gratings, whereas under BonY conditions, the results were within normal limits. Furthermore, comparable to the central retina, scotopic WonW and BonY grating acuities were within normal limits (Fig. 4F, only WonW shown). There was no difference in peripheral grating acuity between the two genotypes in all the conditions tested. The results taken together support the hypothesis that S-cones were likely resolving the gratings in the nasal retina under high photopic conditions in both genotypes of BCM patients. 

Discussion

The molecular complexity of the L- and M-cone opsin genes (OPN1LW and OPN1MW) has been explored not only to understand the basis of color vision but also to elucidate the molecular genetic causes of X-linked cone diseases.^{1,3,6,9,10,11,26–29} BCM causes decreased L/M-cone vision due to simultaneous OPN1LW and OPN1MW gene defects as a result of one of multiple mutation mechanisms, such as large deletions involving upstream regulatory sequences (i.e., locus control region and promoters) and/or parts of or the entire OPN1LW and OPN1MW genes, missense mutations or nonsense mutations, and L/M interchange mutations.^{7,9,26,30–32} Among the more common genotypes reported are large deletions and the C203R missense mutation. Concern has been raised that the mechanism of disease resulting from the missense C203R mutation could involve misfolded cone opsins with potential for a toxic effect on the photoreceptors, comparable to some missense mutations in rhodopsin.^33,34 It is of interest that two individuals with red/green color deficiency and molecular evidence of no OPN1LW genes but a C203R mutation encoded by one of the OPN1MW genes in the photopigment arrays had disruption of the cone photoreceptor mosaic, and this was postulated to cause early degeneration of some of the cone cells but with retained normal acuity.³³ 

There is no consensus in the literature about the level of severity of the BCM phenotype associated with the C203R genotype.^{6,11,27,30,31} Based on the hypothesis of a possible toxic effect of the missense mutation and a more aggressive cone disease in such patients,⁶ we studied patients with the C203R genotype and compared the results with a cohort of patients of similar age range but with large deletion mutations. We previously have demonstrated that in human BCM with deletion mutations there are sufficient residual foveal L/M-cones to warrant a gene therapy approach.¹⁰ In addition, there has been recent proof-of-concept of gene augmentation therapy delivered subretinally in the Opn/mw^−/− mouse mutant.³⁵ The step we took toward understanding the common C203R missense mutation was to ask if the human phenotype differed dramatically from that of patients with deletion mutations. A necessary parallel step would be to develop and characterize a murine model of the C203R mutation and then perform proof-of-concept experiments as was done with the knockout and make that comparison. 

The unexpected result in the present work was that disease progression in patients with the C203R genotype appeared less aggressive than that in our cohort of patients with deletion mutations. There was a definite difference in the foveal cone photoreceptor cell layer thickness across the age spectrum studied in cross-sectional data bolstered by limited longitudinal data. The prolonged period of foveal ONL retention extended over approximately 3 decades of life (from approximately ages 20–50 years) in the patient cohort with the C203R mutation versus the cohort with large deletion mutations. Older patients of both genotypes converged to the same advanced stage of foveal cone loss in their seventh decade of life. This may represent a difference in rates of progression of the two genotype groups or, alternatively, a later onset but similar rate of progression in C203R versus deletion mutation groups. 

It could be asked why a simple and traditional outcome of vision, such as visual acuity, did not show a comparable difference in progression rates between genotypes. At the earliest ages studied in both cohorts of patients, there is a lack of a foveal bulge, a clinical marker for reduced foveal cone OS 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 disruption becomes more complex with age but it is likely that the cone OS abnormality from early life is at least one reason for reduced acuity, no matter how much further damage is incurred from the advancing disease process. Further, in keeping with the reduced acuity throughout the disease course, we have previously used chromatic stimuli and fundus perimetry to position the target on the anatomical fovea in BCM patients mainly with the deletion genotype. The results indicate that almost all of these patients rely on S-cones and rods for perception¹¹ with rare exception.¹⁰ It would therefore not be expected to have the progression rate of foveal L/M-cone structure abnormalities be reflected in the function of these other receptor types. In the current study, a comparison was made of the spatial resolving ability between the two genotypes, using chromatic gratings under fundus viewing. There was no difference in results between the two genotypes. 

What are the clinical trial implications of this observed difference in rate of foveal structural changes between genotypes? The inclusion of BCM patients with the C203R missense mutation in a gene augmentation trial still awaits proof-of-concept experimental results, such as have been reported for a model of deletion mutations, namely, the knockout mouse.³⁵ Also, confirmation of the current human findings with serial data of foveal photoreceptor structural preservation until late in life in BCM with the C203R genotype would be important. A natural history study extending decades is obviously not practical, but longitudinal OCT measurements in a cohort of BCM patients in their sixth or seventh decade of life should be feasible. The present findings suggest that there is a much wider window of opportunity to treat BCM patients with the C203R missense mutation than there may be with the large deletion genotype. 

Acknowledgments

Supported by BCM Families Foundation. 

Disclosure: A. Sumaroka, None; A.V. Garafalo, None; A.V. Cideciyan, None; J. Charng, None; A.J. Roman, None; W. Choi, None; S. Saxena, None; V. Aksianiuk, None; S. Kohl, None; B. Wissinger, None; S.G. Jacobson, None