Rod and Rod-driven Function in Achromatopsia and Blue Cone Monochromatism

Abstract

Purpose

To evaluate rod photoreceptor and postreceptor retinal function in pediatric patients with achromatopsia (ACHR) and blue cone monochromatism (BCM) using contemporary electroretinographic (ERG) procedures.

Methods

Fifteen patients (age 1 to 20 years) with ACHR and six patients (age 4 to 22 years) with BCM were studied. ERG responses to full-field stimuli were obtained in scotopic and photopic conditions. Rod photoreceptor (S_rod, R_rod) and rod-driven postreceptor (log σ, V_max) response parameters were calculated from the a-wave and b-wave. The ERG records were digitally filtered to demonstrate the oscillatory potentials (OPs); a sensitivity parameter, log SOPA_1/2, and an amplitude parameter, SOPA_max, were used to characterize the OP response. Response parameters were compared to those of 12 normal control subjects.

Results

As expected, photopic responses were non-detectable in patients with ACHR and BCM. In addition, mean scotopic photoreceptor (R_rod) and postreceptor (V_max and SOPA_max) amplitude parameters were significantly reduced compared to those in normal controls. The flash intensity required to evoke a half maximum b-wave amplitude (log σ) was significantly increased.

Conclusions

The results of this study provide evidence that deficits in rod and rod mediated function occur in the primary cone dysfunction syndromes, achromatopsia and blue cone monochromatism.

Achromatopsia refers to a group of congenital, stationary retinal disorders in which there is an absence or paucity of functioning cones.^1–3 Complete achromatopsia (ACHR), also called rod monochromatism, is an autosomal recessive condition characterized by reduced visual acuity, photophobia, nystagmus, deficits in color discrimination, and paradoxical pupillary constriction to dark.^1–5 Hyperopia is common ^{1, 6, 7}, although a broad distribution of refractive errors has been reported.⁸ Fundus appearance is typically normal ^1–5, although exceptions have been reported.^{9, 10} Blue cone monochromatism (BCM) is an X-linked condition that shares many of the characteristics of autosomal recessive achromatopsia, sometimes exhibited with reduced severity.^{1–3, 11, 12} Refractive error, however, is typically myopic.^{8, 11, 13–15} Clinically, the Berson plates discriminate patients with BCM from patients with ACHR.^16–18

In ACHR, rods are the only functional photoreceptor type, while in BCM, both rods and short wavelength sensitive cones are functional.^{12, 19} ACHR and BCM are typically regarded as stationary conditions, but in both there have been reports of adults with progressive retinal disease.^{10, 20–26}

Achromatopsia is understood to be a channelopathy of the cone photoreceptors. The most common molecular causes are mutations in the cGMP-gated cation channel genes CNGA3 (OMIM600053) and CNGB3 (OMIM605080).^{7, 27–31} Less frequently, a mutation in the transducin protein GNAT2 (OMIM139340) has been associated with achromatopsia. ^{32, 33} The most common molecular causes of BCM are mutations in the opsin gene array of long and medium wavelength sensitive cone visual pigments located adjacently on the X-chromosome. (OMIM303700).^{25, 34}

In both ACHR and BCM, cone and cone-driven electroretinogram (ERG) responses to full-field stimuli are markedly attenuated or non-detectable, whereas rod and rod-driven responses are typically reported to be normal or near normal.^{4, 6, 8, 9, 21, 26, 27, 35–40} Recently, however, abnormal rod-driven ERGs have been reported in some patients with CNGB3 achromatopsia ¹⁰ and BCM.²³

Our own clinical observations also indicated abnormalities in rod and rod-driven ERGs in pediatric patients with achromatopsia and blue cone monochromatism. Therefore, we undertook an analysis of rod photoreceptor and postreceptor ERG components. Our goal was to identify possible mechanisms underlying the abnormalities.

Methods

Subjects

Twenty one patients (Table 1), 15 with complete achromatopsia (ACHR) and six with blue cone monochromatism (BCM), who had been followed in the Department of Ophthalmology, Children’s Hospital Boston were studied retrospectively. ACHR patients exhibited typical features of achromatopsia, including low visual acuity; photophobia; low amplitude, high frequency, “jelly-like” nystagmus; and paradoxical pupillary constriction to dark. All patients had normal fundus appearance. ACHR patients #1 and #8 are siblings. The clinical presentation of the patients with BCM was similar, although the photophobia often appeared less severe. All were male and all passed the Berson test;¹⁶ that is, unlike patients with ACRH, they were able to distinguish a purple-blue (Munsell Color System 7.5 PB; dominant wavelength 468 nm) arrow from blue-green (5.0 BG; 491 nm) arrows. Two males who were classified as ACHR (ACHR #4 and #7) were too young to do color vision testing. Median age at ERG was 2.7 years (range 1 to 20 years) for the ACHR patients and 8.0 years (range 4 to 22 years) for the BCM patients.

Table 1.

Characteristics of Achromatopsia and Blue Cone Monochromatism Patients

Patient No	Sex	Age at ERG (yr)	Spherical Equivalent (D)	Visual Acuity (most recent)	Photophobia	Nystagmus	PPR*
ACHR
1	F	1.0	+3.75‡	20/190 §	Yes	Yes	Yes
2	F	1.1	+5.50‡	20/960 §	No	Yes	Yes
3	F	1.2	+6.50‡	20/250	Yes	Yes	No
4	M	1.5†	+7.75‡	20/500	Yes	Yes	No
5	M	1.6	+0.25	20/125	Yes	Yes	Yes
6	F	2.3	+4.00‡	20/133§	Yes	Yes	Yes
7	M	2.3	+8.50‡	20/180§	Yes	Yes	No
8	M	2.7	+2.00	20/400	Yes	Yes	Yes
9	F	3.0	+4.25‡	20/250	Yes	Yes	Yes
10	F	3.9	+8.00‡	20/300	Yes	Yes	Yes
11	F	4.9	−3.00‡	20/320	Yes	Yes	Yes
12	M	6.2†	+3.75‡	20/200	No	Yes	No
13	M	7.0	+1.00	20/222	Yes	Yes	Yes
14	M	7.7†	−4.75‡	20/200	Yes	Yes	No
15	F	20.2†	−1.50	20/125	Yes	Yes	Yes
BCM
16	M	4.0	−2.88‡	20/160	Mild	Yes	Yes
17	M	6.8	−1.75‡	20/209	No	Yes	Yes
18	M	7.7	−6.13‡	20/400	Yes	Yes	Yes
19	M	8.3†	−1.63	20/160	Mild	Yes	No
20	M	9.4†	−0.50	20/80	Mild	Yes	Yes
21	M	22.1†	−10.25‡	20/130	Mild	Yes	No

^*PPR: paradoxical pupillary response

^†tested awake

^‡outside 99% prediction limit for normal⁴¹, ⁴²

^§grating acuity (Teller Acuity Cards)⁹⁷

Visual acuity was measured in dim room light using age appropriate tests (Teller Acuity Cards, HOTV, Lea, Feinbloom, or ETDRS). Refractive error was measured using cycloplegic retinoscopy.⁴¹ The most recent visual acuity and spherical equivalent values for each patient are reported in Table 1. Acuity was below normal for age in all patients. Twelve of the 15 ACHR patients were hyperopic; nine were outside the 99% prediction limit of normal for age.^{41, 42} Two of the three myopic ACHRs were also outside the normal limit. All six BCM patients were myopic; four were outside the 99% prediction limit of normal for age.

Dark adapted, rod mediated visual thresholds, obtained in 11 ACHR patients and five BCM patients, were normal ⁴³ in all but one (ACHR #7), who showed a mild (1.18 log units) but statistically significant threshold elevation. Four patients had repeated measurements with a 1.5 year (ACHR #5), a 6.8 year (BCM #21), a 7.8 year (ACHR #9), and a 9.0 year (ACHR #12) interval between tests, and none showed a change in threshold, suggesting a stationary condition.

ERG responses in the patients with ACHR and BCM were compared to responses in 12 healthy control subjects (median age 23 years; range 8 to 41 years). ERG response parameters in normal subjects at the ages of our patients with ACHR and BCM (1 to 22 years) do not differ significantly from those in adults.⁴⁴ All of the control subjects had normal ocular structures and corrected visual acuity of 20/25 or better; median spherical equivalent was −0.50 D (range −4.75 to +0.88 D).

The patients had been referred for examination and testing in an attempt to diagnose their eye and vision problems. Analysis of the patients’ data was done retrospectively with the approval of the Children’s Hospital Committee on Clinical Investigation (CCI). Written, informed consent was obtained from the control subjects after explanation of the nature and possible consequences of the study. The control study conformed to the tenets of the Declaration of Helsinki and was approved by the Children’s Hospital CCI.

Electroretinography

Pupils were dilated with 1% cyclopentolate hydrochloride, and the patient was dark adapted for 30 minutes. All 12 control subjects and seven patients (four ACHR, three BCM) were tested awake (Table 1); fifteen patients (11 ACHR, three BCM) had ERG testing under light inhalation anesthesia which has no significant effect on the ERG parameters studied herein.⁴⁵ Following dark adaptation, 0.5% proparacaine was instilled and, under dim red light, a bipolar Burian-Allen electrode (Hansen Ophthalmic Development Laboratory, Coralville, IA) was placed on the cornea. A ground electrode was placed on the skin over the mastoid. Responses were recorded from both eyes of the patients and from one eye of the control subjects. In patients, the eye with the larger scotopic amplitudes was selected for analysis.

Thirteen of the patients (10 ACHR, three BCM) and all 12 control subjects were tested using a Compact 4 system (Nicolet, Madison, WI) and eight (five ACHR, three BCM) using an Espion system (Diagnosys, Lowell, MA). Despite differences between the two recording systems in the spectral composition of the stimuli (described below) and in data acquisition (2,564 Hz digitization rate for the Nicolet; 2,000 Hz for the Espion), a previous comparison ⁴⁶ of rod and cone photoresponse parameters in normal adult subjects obtained using the Espion system (N=7) and obtained earlier using the Nicolet system (N=13) ^{44, 47} showed no significant differences. Therefore, the data obtained using the two systems have been combined.

Responses were differentially amplified, displayed, digitized, and stored for analysis. A voltage window was used to reject responses contaminated by artifacts. Two to 16 responses were averaged in each stimulus condition. The inter-stimulus interval ranged from 2 to 60 seconds and was selected so that subsequent b-wave amplitudes were not attenuated.⁴⁷

Full-field stimuli were presented in an integrating sphere. Stimulus intensity was measured using a calibrated photodiode (IL1700; International Light, Newburyport, MA) placed at the position of the subject’s cornea. The troland values of the stimuli were calculated by taking each subject’s pupillary diameter into account. To test rod function, after dark adaptation, responses to brief (<3 ms), short wavelength stimuli ranging from those that evoked a small b-wave (<15 μVolts) to those saturating the a-wave were recorded. In the Nicolet system, a Wratten 47B filter (λ<510 nm) was used; in the Espion system, a 470 nm LED (half bandwidth 30 nm) was used. Flashes were presented over a >4 log unit range, starting with the dimmest and increasing in 0.3 log unit steps. The maximum intensity flash produced approximately 3.0 log scotopic troland seconds (scot td sec) for an 8 mm pupil.

To isolate rod function in the control subjects, dark adapted responses to photopically matched long wavelength flashes (Wratten 29 filter, λ>610 nm) were recorded and subtracted from the responses to the corresponding short wavelength flashes.⁴⁸

Cone function was tested using long wavelength flashes. In the Nicolet system, a Wratten 29 filter (λ >610 nm) was used; in the Espion system, a 630 nm LED (half bandwidth 30 nm) was used. A 1.8 log unit range of red flash intensities was presented on a steady, rod-saturating background (~3 log phot td). The maximum intensity flash produced approximately 3.2 log photopic troland seconds (phot td sec) for an 8 mm pupil. Seventeen of the 21 patients were also tested with a 30 Hz flickering white stimulus (2.4 log phot td sec).

The rod photoresponse characteristics were estimated from the a-wave by means of the Hood and Birch formulation ⁴⁹ of the Lamb and Pugh model ^50–52 of the biochemical processes involved in the activation of rod phototransduction. A curve fitting routine (MATLAB, fmins subroutine; The MathWorks, Inc, Natick, MA) was used to determine the best fitting values of S_rod [(scot td)⁻¹ sec⁻³], R_rod (μVolts), and t_d (a brief delay, sec) in the following equation:

$$\text{P}3(\text{I},\text{t})=\{1-exp[-0.5\text{I}{\mathbf{S}}_{\mathbf{rod}}{(\text{t}-{\mathbf{t}}_{\mathbf{d}})}^2]\}{\mathbf{R}}_{\mathbf{rod}}\text{for}\text{t}>{\mathbf{t}}_{\mathbf{d}}$$ Eq. 1

In this equation, I is the flash in scotopic troland seconds. Assuming that the number of isomerizations of rhodopsin produced by the stimulus is known, the term S_rod is related to the amplification constant, A, in the molecular models.^50–53 In these models, A summarizes the kinetics of the series of processes, initiated by the photoisomerization of rhodopsin, that result in closure of the channels in the plasma membrane of the photoreceptor. R_rod is an estimate of the amplitude of the saturated response. Fitting of the model was restricted to the leading edge of the a-wave or to a maximum of 20 ms after stimulus onset.

The b-wave responses to short wavelength flashes were also analyzed. The stimulus/response function

$$\text{V}(\text{I})={\mathbf{V}}_{\mathbf{max}}[\text{I}/(\text{I}+\sigma )]$$ Eq. 2

was fit to the b-wave amplitudes of each subject. In this equation, V is the b-wave amplitude produced by flash intensity I, V_max (μVolts) the saturated amplitude, I the stimulus in scot td sec, and σ the stimulus that evokes a half-maximum b-wave amplitude. The function was fit only up to those higher intensities at which substantial a-wave intrusion occurred (~+1.0 log scot td sec).⁵⁴

As established by Granit, the ERG waveform represents the algebraic sum of photoreceptor and postreceptor retinal responses.^{55, 56} The isolated rod photoresponse, called P₃, is modeled by Equation 1. To evaluate postreceptor function, designated P₂, a putatively “pure” postreceptor response was isolated from the intact ERG by digital subtraction of P₃ from the record. P₂ represents primarily the bipolar cell response.^57–61

For P₂, the relation between flash intensity and the elapsed time between stimulus presentation and the instant at which the response reaches an arbitrary criterion voltage will be linear on a log-log plot with slope −0.2 in normal retina, consistent with three stages of integration in the rod photoreceptor and three stages of integration in the rod bipolar cell.⁵⁹ Departures from this relation indicate dysfunction of the ON bipolar cells’ G-protein cascade.^{59, 60} We selected a 50 μVolt criterion and noted the latency at which the rising phase of P₂ reached that criterion. For a family of P₂ waves, we plotted the latency versus intensity relationship. To test for dysfunction, regression lines were fit and the slope of the regression line (P₂ slope) in patients was compared to that in control subjects.

Oscillatory potentials (OPs) were extracted from the derived postreceptor response (P₂) as described previously.⁶² In brief, P₂ was digitally filtered using a fifth-order Butterworth filter (MATLAB; butter subroutine; The MathWorks, Inc, Natick, MA) with bandpass 75 to 300 Hz.⁶³ The amplitude (μV) of each OP wavelet was defined as the difference between the peak and the trough immediately preceding it. To characterize the OPs, the summed amplitude of the OPs (SOPA) at each intensity was plotted as a function of stimulus energy and the Michaelis-Menton equation

$$\text{SOPA}(\text{I})=\mathbf{SOP}{\mathbf{A}}_{\mathbf{max}}[{\text{I}}^{\text{n}}/({\text{I}}^{\text{n}}+\mathbf{SOP}{{\mathbf{A}}_{1/2}}^{\text{n}})]$$ Eq. 3

was fit to the data. In this equation, SOPA(I) is the summed amplitude (μV) of the OPs in the response to a flash of I intensity, SOPA_max is the saturated amplitude (μV) of the OPs, and SOPA_½ is the intensity at which the summed amplitude of the OPs is half SOPA_max.

Statistical Analyses

Preliminary analyses showed no significant difference between ACHR and BCM patients on any of the ERG parameters (S_rod, R_rod, log σ, V_max, log SOPA_1/2, SOPA_max, and P₂ slope; t-tests: df=19; P >0.2 on all tests). Furthermore, for each parameter, the range of values for ACHR and BCM patients was similar. Therefore, data from the two patient groups were pooled and individual, independent samples t-tests for each ERG parameter were used to detect differences between patients and control subjects. The significance level for all tests was P<0.01.

Results

In Figure 1, sample ERG records from an ACHR patient obtained in scotopic (Fig. 1A) and photopic (Fig. 1B) conditions are shown. In all patients with ACHR or BCM, scotopic activity was observed over a ≥3 log unit range of intensities, while photopic activity was absent or markedly attenuated (<5% of normal mean amplitude). Figure 1C shows sample fits of the model (Eq. 1) of the activation of rod phototransduction ^49–52 to the a-wave. Figure 1D shows the fit of Eq. 2 for determining the postreceptor (b-wave) response parameters. Note that lower values of log σ indicate greater sensitivity; that is, a lower intensity produces the half maximum response.

Figure 1.

Sample ERG results from an achromatopsia patient (ACHR #9) whose scotopic amplitudes are near the mean for patients. A. Dark-adapted ERG responses to a series of short wavelength flashes. For clarity, the stimulus intensity in log scot td sec is shown only for every other trace. B. Light-adapted ERG responses to two long wavelength flash intensities, +3.2 and +2.4 log phot td sec, and to 30 Hz flickering white light (+2.4 log phot td sec). The calibration bar pertains to panels A and B. C. The first 40 milliseconds of the ERG (solid lines) and the fit of Eq. 1 (dashed lines) to the leading edge of the a-wave. The parameters S_rod and R_rod for the model fit to the data are indicated. D. B-wave amplitude plotted as a function of stimulus intensity. Eq. 2 was fit up to ~+1.0 log scot td sec, indicated by the closed circles; parameters V_max and log σ are indicated.

Figure 2 shows P₂ responses derived from the records in Figure 1A and a plot of the latency versus intensity relationship. Scotopic OP records extracted from the P₂ responses shown in Figure 2 are displayed in Figure 3; OPs from a control subject are also shown.

Figure 2.

A. Sample P₂ records from ACHR #9. The derivation of P₂ is shown in the inset. B. Latency at the 50 μVolt criterion plotted as a function of log stimulus intensity. The regression line has a slope of −0.20.

Figure 3.

Scotopic oscillatory potentials for a series of flash intensities from ACHR #9 (left) and for a control subject (right). For clarity, the stimulus intensity in log scot td sec is shown only for every other trace. Note that the achromatopsia patient’s records are plotted at twice the gain of the control subject’s.

The rod photoreceptor and postreceptor response parameters for the patients with ACHR and BCM and for control subjects are summarized in Figure 4. The amplitude parameters for the rods, R_rod (t = −7.484, df=31, P<0.001), and for postreceptor activity, V_max (t = −6.821, df=31, P<0.001) and SOPA_max (t = −10.755, df=31, P<0.001), were significantly lower in patients than in controls, with little overlap. For all sensitivity parameters (S_rod, log σ, and log SOPA_1/2), there was substantial overlap between patients and controls. However, b-wave log σ in patients differed significantly from that in controls (t = 3.152, df = 31, P = 0.0036); in patients, a higher intensity was needed to evoke a half maximum response. In prior study of young, healthy subjects with myopia (as high as −10 diopters), we found no differences in rod and rod-driven postreceptor response parameters between myopes and controls.⁶⁴

Figure 4.

Rod photoresponse parameters, S_rod and R_rod, and postreceptor parameters, log σ, V_max, log SOPA_1/2, and SOPA_max. Amplitude parameters (R_rod, V_max, and SOPA_max) are shown on the top and sensitivity parameters (S_rod, log σ, and log SOPA_1/2) on the bottom. Log values are plotted for all parameters. Data for control subjects (triangles), patients with ACHR (filled circles), and BCM (open circles) are shown in each graph. The horizontal lines indicate the mean for each group.

The mean slope of regression lines fit to P₂ latency versus intensity plots was −0.19 (SD=0.04) in the 12 control subjects and −0.20 (SD = 0.04) in the ACHR and BCM patients. These values were not significantly different from each other or from the normal mean slope of −0.2.^{59, 60, 65}

Discussion

In these young patients with achromatopsia (ACHR) and blue cone monochromatism (BCM), we have demonstrated significant deficits in rod and rod-driven function. Specifically, mean photoreceptor (R_rod) and postreceptor (V_max and SOPA_max) amplitude parameters were reduced relative to those in normal controls (Figure 4). Sensitivity parameters (S_rod, log σ, and log SOPA_1/2) were less affected; only b-wave log σ differed significantly from normal.

While rod photoreceptors are not directly affected by the genetic mutations causing ACHR and BCM, it has been suggested that alterations in rod structure occur. High resolution adaptive optics imaging of the photoreceptor mosaic in a subject with CNGB3 achromatopsia showed increased diameter of rod inner segments, possibly due to rods expanding into space that would normally be occupied by cones.⁶⁶ In this subject, the density of rods at 10° eccentricity was reduced by about a third compared to normal.^{66, 67} Thus, the low values of R_rod in our ACHR and BCM subjects may be due to a decrease in the total number of rods. Shorter rod outer segment length would also reduce R_rod. To our knowledge, rod outer segment length has not been measured in ACHR or BCM.

To our knowledge, only one other study ¹⁰ quantitatively investigated rod activation in patients with ACHR. Khan and colleagues evaluated four adults with CNGB3 achromatopsia who showed macular atrophy in middle age. Their rod photoreceptor and postreceptor amplitude parameters fell within the range of values observed in our patients. Our patients, however, were younger than theirs (Table 1) yet the majority showed greater deficits. We have observed neither fundus abnormalities nor progressive worsening in visual acuity or dark adapted visual thresholds in our patients. We wonder, therefore, if the alterations in rod and rod-driven function may indicate anomalies in rod pathway signaling rather than rod disease. Individuals with ACHR and BCM prefer dim environments, which would increase the metabolic load placed on the rods. This, in turn, would result in more circulating current, which would require more energy with possible adverse long term effects on rod function.

Thus, the low calculated values of R_rod could be due to fewer rods, shorter rod outer segments, or defective rod functioning.

In addition to the significant deficit in rod photoresponse amplitude, we observed deficits in postreceptor response parameters (V_max, log σ, and SOPA_max). According to an explicit model, changes in R_rod are predicted to alter b-wave sensitivity (log σ) but to have little effect on V_max.58, 68 In our patients, mean log σ and mean V_max are both approximately half that in controls. The low V_max could be caused by too few rod-driven bipolar cells. Although we are unaware of any anatomic evidence that the number of rod bipolar cells is reduced in ACHR or BCM, the reduced rod density found in the subject with achromatopsia ⁶⁶ may be accompanied by a proportionate reduction in rod bipolar cell density. In another system (immature simian central retina), the numbers of cones and cone bipolar cells are proportionately decreased.⁶⁹ Another possible explanation for the reduction in V_max that is consistent with the explicit model ^{58, 68} is a postreceptor change resulting from abnormal function of rod bipolar cells. However, the normal P₂ latency versus intensity slope (Figure 2) indicates that, at the least, the G-protein amplification cascade in the rod bipolar cell was operational.

Our data do not allow us to exclude the possibility that there is some alteration of the rod-driven circuitry in ACHR and BCM. Reorganization of the postreceptor retina is a well documented consequence in a number of photoreceptor disorders.^{62, 70–73} The normal scotopic pathway is dominated by the rod-specific hyperpolarizing bipolar cell.⁷⁴ In addition to this primary pathway, there are anatomical connections between rods and cones and some between rods and depolarizing cone bipolar cells.^75–81 We speculate that the latter contacts may be more numerous in cone-deficient ACHR and BCM retinas. This would allow substantial rod input to cone depolarizing bipolar cells with consequent reduction in the apparent postreceptor response from the primary rod pathway in both ACHR and BCM. In a CNGA3^−/− mouse model, anomalous synapses between rods and cone bipolar cells are documented.⁸²

The oscillatory potentials are affected by inputs from both rods and cones.^{83, 84} In ACHR and BCM retinas, cone input is absent or greatly diminished, possibly accounting for the dramatic attenuation in SOPA_max observed in our patients.

Whatever the actual mechanisms, the ERG data reported herein add evidence that deficits in rod and rod mediated function occur in the primary cone dysfunction syndromes, achromatopsia and blue cone monochromatism. While it is well established that cones are adversely affected in primary rod disorders,^{82, 85–91} there has been less evidence that rods are affected in disorders with primary cone dysfunction.^{10, 23, 92}

Each of the possible mechanisms for abnormal retinal function considered above leads to hypotheses that can be tested by further ultra-high resolution imaging of individuals with ACHR and BCM and by study of animal models.^{82, 92–94} The new knowledge obtained will bolster efforts to design and evaluate effective therapies for cone dysfunction syndromes.^{95, 96}