August 2012
Volume 53, Issue 9
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Eye Movements, Strabismus, Amblyopia and Neuro-ophthalmology  |   August 2012
Characterization of Pupil Responses to Blue and Red Light Stimuli in Autosomal Dominant Retinitis Pigmentosa due to NR2E3 Mutation
Author Affiliations & Notes
  • Aki Kawasaki
    From the Neuro-Ophthalmology Unit, Hôpital Ophtalmique Jules Gonin, University of Lausanne, Lausanne, Switzerland;
    Department of Clinical Sciences and Ophthalmology, University of Umeå, Umeå, Sweden;
  • Sylvain V. Crippa
    From the Neuro-Ophthalmology Unit, Hôpital Ophtalmique Jules Gonin, University of Lausanne, Lausanne, Switzerland;
  • Randy Kardon
    Department of Ophthalmology and Visual Science, University of Iowa, and Center for the Prevention and Treatment of Visual Loss, Department of Veterans Affairs Medical Center, Iowa City, Iowa; and
  • Lorette Leon
    From the Neuro-Ophthalmology Unit, Hôpital Ophtalmique Jules Gonin, University of Lausanne, Lausanne, Switzerland;
  • Christian Hamel
    Genetic Sensory Disease, Department for Rare Diseases, University Hospital of Montpellier and INSERM U1051, Montpellier, France.
  • Corresponding author: Aki Kawasaki, Hôpital Ophtalmique Jules Gonin, Avenue de France 15, Lausanne, Switzerland 1003; aki.kawasaki@fa2.ch.  
Investigative Ophthalmology & Visual Science August 2012, Vol.53, 5562-5569. doi:10.1167/iovs.12-10230
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      Aki Kawasaki, Sylvain V. Crippa, Randy Kardon, Lorette Leon, Christian Hamel; Characterization of Pupil Responses to Blue and Red Light Stimuli in Autosomal Dominant Retinitis Pigmentosa due to NR2E3 Mutation. Invest. Ophthalmol. Vis. Sci. 2012;53(9):5562-5569. doi: 10.1167/iovs.12-10230.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: We characterized the pupil responses that reflect rod, cone, and melanopsin function in a genetically homogeneous cohort of patients with autosomal dominant retinitis pigmentosa (adRP).

Methods.: Nine patients with Gly56Arg mutation of the NR2E3 gene and 12 control subjects were studied. Pupil and subjective visual responses to red and blue light flashes over a 7 log-unit range of intensities were recorded under dark and light adaptation. The pupil responses were plotted against stimulus intensity to obtain red-light and blue-light response curves.

Results.: In the dark-adapted blue-light stimulus condition, patients showed significantly higher threshold intensities for visual perception and for a pupil response compared to controls (P = 0.02 and P = 0.006, respectively). The rod-dependent, blue-light pupil responses decreased with disease progression. In contrast, the cone-dependent pupil responses (light-adapted red-light stimulus condition) did not differ between patients and controls. The difference in the retinal sensitivity to blue and red stimuli was the most sensitive parameter to detect photoreceptor dysfunction. Unexpectedly, the melanopsin-mediated pupil response was decreased in patients (P = 0.02).

Conclusions.: Pupil responses of patients with NR2E3-associated adRP demonstrated reduced retinal sensitivity to dim blue light under dark adaptation, presumably reflecting decreased rod function. Rod-dependent pupil responses were quantifiable in all patients, including those with non-recordable scotopic electroretinogram, and correlated with the extent of clinical disease. Thus, the chromatic pupil light reflex can be used to monitor photoreceptor degeneration over a larger range of disease progression compared to standard electrophysiology.

Introduction
Autosomal dominant retinitis pigmentosa (adRP) is a phenotypically and genetically heterogeneous disorder. To date, specific mutations in 24 genes have been identified as pathogenic for this condition. 13 A heterozygous transition c166G > A in exon 2 of the NR2E3 gene results in a missense mutation Gly56Arg, which leads to clinical adRP. 35 The typical phenotype is a progressive rod-cone dystrophy. 5  
In most retinal degenerative disorders, including NR2E3-associated adRP, there comes a point in the evolution of the disease when rod and cone responses derived from the electroretinogram (ERG) are barely detectable or completely extinguished, yet the patient still has subjective visual perception. This relative insensitivity of the ERG for registering low-level electrical activity from the outer retina is well known. In such patients, residual rod and cone function can be assessed by perimetric tests, 6,7 but such tests demand patient cooperation and attention. As novel interventions are being introduced to treat certain genetically-determined retinal degenerations, there is renewed interest in alternative means to define a greater dynamic range of rod and cone function. Quantification of the pupil light reflex is one such alternative test and has the appeal of being a noninvasive technique, and having a relatively rapid testing time and minimal dependency on patient cooperation, such as in young children. 
Early pupillographic works demonstrated that, under selected stimulus conditions, pupil responses can reflect photoreceptor function. 810 Recent works have suggested that the pupil light reflex may be more sensitive for detecting residual rod and cone activity in more severe stages of retinal degeneration compared to standard full-field ERG. 1113 We have used chromatic pupillometry in patients with Leber congenital amaurosis (LCA) who had very poor visual function. 14 Despite absent or negligible rod function, as determined from pupil responses, we found that residual cone function was readily detectable and quantifiable in these patients whose ERG had long ago become non-recordable, lending further support for using the pupil light reflex to monitor outer retinal degenerative disease. 
In this study, we evaluated pupil responses to selective wavelength (colored) light in 9 patients from two families with adRP due to the p.Gly56Arg mutation in the NR2E3 gene. To understand how clinical disease affects the pupil response to blue and red light, we measured the stimulus-response function of the pupil over a range of light intensities under dark- and light-adapted conditions. From analysis of pupil responses in specific stimulus conditions, the rod, cone, and melanopsin function was quantified and compared to other clinical indicators of retinal function. The main aim of our study was to characterize the pattern of pupillographic responses in a genetically homogeneous population to define better the use of the chromatic pupil light reflex in the clinical setting. 
Methods
Patients
This prospective, nonrandomized, interventional design study was conducted according to the tenets of the Declaration of Helsinki, and received authorization from the South Mediterranean IV ethical board committee for human research and from the French regulation agency for medication. All study participants provided oral and written informed consent. Patients were recruited, examined, and tested at the Gui de Chauliac Hospital, Institut des Neurosciences de Montpellier in Montpellier, France. Patients with a clinical diagnosis of adRP carrying the p.Gly56Arg mutation in NR2E3 gene were selected from a genetic sensory disease database and the mutation was confirmed by direct sequencing. 
Charts of affected members in two families were reviewed to exclude those with any other cause of visual loss other than the primary ocular diagnosis of retinal degeneration. Nine patients were available to undergo a standard ophthalmologic examination, which included visual acuity, static perimetry (MonPack1, [STAT-10] 56 points, central 10 deg; Metrovision, Pérenchies, France), automated kinetic perimetry (MonPack1; Metrovision), optical coherence tomography (OCT Stratus, program macular thickness map; Carl Zeiss, Dublin, CA), and an experimental intervention, which was pupil testing with a computerized chromatic pupillometer (see Methods below). The following information was recorded from the hospital chart: date and results of most recent full-field ERG, fundus description, and/or photographic documentation. 
Nine healthy volunteers were recruited from hospital personnel and 2 from accompanying persons of patients. One boy who was the son of one of the investigators (AK) also served as control subject and consent was obtained from the non-investigator parent. These 12 control subjects had a normal ophthalmologic examination and no history of ocular disease other than refractive error, and were not using topical or systemic medications known to affect the pupil light reflex. 
Pupillometry
Computerized pupillography was performed under conditions of dark and light adaptation in the right eye of all study participants. Details of the instrumentation have been described previously. 14,15 In brief, a ColorDome Ganzfeld ERG apparatus (Diagnosys, Lowell, MA) was used to present a full-field 1-second light stimulus at preselected spectral bandwidths of 640 ± 10 nm (red light) and 467 ± 17 nm (blue light). Light intensities used in our study ranged from −6.0 to 2.6 log cd/m2 (0 log = 1 cd/m2). The untested eye was occluded with a patch and the tested (stimulated) eye also was the monitored eye. A dual channel binocular pupillometer mounted on an eye frame (Arrington Research, Scottsdale, AZ) continuously recorded the pupil diameter of the stimulated eye at 30 Hz for the duration of each pupil test (see below). 
We developed 2 pupil tests, modified from a recently reported stimulus protocol, 12 for use in our study. In the first pupil test, the patient was dark-adapted (0 cd/m2) for 10 minutes. The pupil testing sequence began with a 10-second period of total darkness followed by a series of alternating blue and red stimulus lights, starting at a very dim intensity (sub-threshold for visual perception and pupil response) and then increasing by 0.5 log-unit steps over a 7 log-unit range (−6.0 to 1.0 log cd/m2). The dark interval between light stimuli had been determined previously (from 3–30 seconds, with increasing dark intervals at brighter light intensities) to allow the pupil to return to baseline size before the next light stimulation. A bright (2.6 log cd/m2) red and blue stimulus was presented at the end of the test sequence to assess the sustained post-illumination pupil response attributable to melanopsin activation. 
The conditions of the second pupil test were selected to favor cone activation while minimizing the rod and melanopsin contributions to the pupil response. These specifications included pre-test light-adaptation, a continuous short wavelength background light in the Ganzfeld bowl, long wavelength (red) light stimulus, and higher stimulus intensities. The second test was performed following 10 minutes of adaptation to room light and 3 minutes of adaptation to a blue light (0.78 log cd/m2) in the Ganzfeld bowl. A series of 1-second red-light stimuli of increasing intensity (range −1.0 to 1.5 log cd/m2; 0.5 log-unit steps) was presented. In addition, a bright red and blue stimulus (2.6 log cd/m2) was given at the end of the testing sequence. 
The study participants were informed that the stimulus light might appear as white (no color), blue, or red. For each test, the subject was asked to signal (by knocking on the table) when a stimulus light first was seen and also to verbalize the color of the stimulus light when a color (blue or red) was first perceived. 
Main Outcome Measures
The pupil data were exported and analyzed in a spreadsheet (Microsoft Excel 2002, Visual Basic for Applications; Microsoft, Zurich, Switzerland). Blink artifacts were removed from the raw pupil tracings with a customized semi-automated filter function. The baseline pupil size was derived from the median size during the 1 second just before onset of each light stimulus. Percent pupil constriction at each time point was calculated by the following formula: Percent pupil constriction at time x = ([baseline pupil diameter minus pupil diameter at time x]/[baseline pupil diameter]) × 100. The pupil response to a light stimulus was defined as the maximal percent pupil constriction that occurred within the duration of the light stimulus and having at least 500 ms latency from light onset. A criterion level of 5% contraction was applied to distinguish evoked pupil responses from random noise. 
The pupil response was plotted as a function of stimulus light intensity, and a response curve was fitted (GraphPad Prism version 5.00 for Windows; GraphPad Software, San Diego, CA) by using an asymptotic exponential function. This was done for the dark-adapted blue-light, the dark-adapted red-light, and the light-adapted red-light stimulus conditions. 
Specific outcome measures derived from the recorded pupil responses were the response threshold (the lowest intensity generating a pupil light reflex) and the logI50 (intensity generating a half-maximal response). We defined the “threshold” as the lowest stimulus light intensity expected to produce a 5% criterion pupil constriction. We applied a linear regression analysis through pupil responses to the dimmest blue stimuli ( −6.0 to −1.0 log cd/m2). If the subject had more than one null response (no pupil constriction above noise), then only the last null response before the first measured response was included in the regression analysis. Extrapolation of the regression line to the intersect Y = 5, that is where pupil constriction amplitude first attains a 5% criterion level, determined the intensity of the rod-dependent pupil response threshold (hereafter called rod threshold). 12,14 The response threshold of cones (hereafter called cone threshold) was determined from a similar analysis to pupil responses in the light-adapted red-light stimulus condition. 
LogI50 is the intensity that produces a pupil response that is half of the maximal response for the given stimulus light condition. The logI50 of the dark-adapted blue-light and the light-adapted red-light stimulus conditions serves as a general measure of the sensitivity of rods and cones, respectively. The functional interaction between rods and cones, however, was assessed by calculating the difference in retinal sensitivity (logI50) to equivalent blue and red light stimuli presented under the same testing condition of dark adaptation. 
Persistence of pupillary constriction after termination of a bright blue light stimulus is the characteristic pupillographic feature of intrinsic melanopsin activation. 12 Our red stimulus wavelength is at the limit of the melanopsin spectral sensitivity curve and, in our study, red light did not evoke a sustained pupil constriction. The red stimulus (2.6 log cd/m2) thus served as a control stimulus for comparing the pupil response to a photopically matched bright blue light. We defined operationally intrinsic melanopsin activity as the difference between the relative pupil size at 6 seconds after termination of the red light and the relative pupil size at 6 seconds after termination of the blue light. 
From the ERG data, rod and cone function was estimated from the b-wave amplitude (in μV) to −25 decibels (dB) white light under dark adaptation and to 0 dB white light under light-adaptation (ISCEV standards), respectively. The automated kinetic visual field (KVF) was quantified objectively using a manual dot overlay technique that assesses the area contained within the III4 isopter and assigns a number between 0 and 100, with 100 being the best possible score. 16,17 For each patient, the extent of clinical expression of the disease was assessed from the combination of findings of the clinical examination, ERG, KVF, and static perimetry, and then patients were ranked from 1 to 9, with 1 demonstrating the mildest disease. 
Statistics
Descriptive statistics were used to characterize the distribution of the rod and cone thresholds, the sensitivity of the pupil light reflex to blue and red light (logI50 for dark-adapted blue, dark-adapted red, and light-adapted red-light stimulus conditions), and the thresholds for visual perception in the control group. The distribution of these parameters for patients was analyzed and compared to the control group using paired group analysis. Spearman rank correlation analyses were performed to examine for a relationship between the quantified pupil parameters (threshold and retinal sensitivity) and the extent of clinical disease. 
Results
The control group comprised 12 subjects (6 men and 6 women) aged 12 to 57 years. The 9 patients represented two families having the same p.Gly56Arg mutation of the NR2E3 gene. They were 6 men and 3 women, aged 13 to 60 years. A clinical description of each patient is summarized in Table 1. The extent of clinical expression of disease ranged from mild to advanced and correlated with increasing age (r 2 = 0.7, P < 0.05), but not with any other clinical parameter. The patients had visual acuity that ranged from 0.5 to 1.0 and a KVF score from 0 to 100. Eight patients had available ERG data (Table 1). Five patients had a non-recordable rod response and 3 patients had severe reduction of the scotopic b-wave amplitude to 30% of normal amplitude or less. Two of the 5 patients with non-recordable scotopic ERG also had no detectable cone response. In 6 patients, the cone ERG response was mildly-to-moderately reduced (45%–78% of normal). 
Table 1. 
 
Clinical Features of Nine Patients with NR2E3 Gene Mutation (p.Gly56Arg)
Table 1. 
 
Clinical Features of Nine Patients with NR2E3 Gene Mutation (p.Gly56Arg)
Patient by Rank and Family (A or B) Age (y) / Sex (M/F) Symptoms VA Fundus CVF Defect (MD in dB) KVF Score Scotopic ERG (% of Normal −25 dB B-Wave Amplitude) Photopic ERG (% of Normal 0 dB B-Wave Amplitude )
P1 (B) 13/M None 1.0 Few atrophic areas in mid periphery, pigment clump inferotemporal periphery −0.9 100 27% 78%
P2 (A) 21/M None 1.0 Peripapillary depigmentation −0.1 87 30% 45%
P3 (B) 28/M Night blindness 0.7 Normal fundus 14.6 92 Not done Not done
P4 (B) 16/F Night blindness 1.0 Pigment atrophy in peripapillary and mid peripheral areas 13.4 51 0% 70%
P5 (A) 47/M Night blindness, photophobia 0.5 Atrophic perifoveal ring, pigment clumps in temporal periphery 13.1 70 0% 55%
P6 (B) 42/F Night blindness, photophobia 0.7 White deposit along vascular arcades, few pigment clumps in mid periphery 16.5 25 26% 54%
P7 (A) 40/F Night blindness 1.0 Normal fundus 0 28 0% 70%
P8 (B) 60/M Night blindness 0.5 Many bone-spicules in the mid periphery, pigment clumps in extreme periphery, attenuated vessels 18.3 0 0% 0%
P9 (A) 44/M Night blindness, mild photophobia 0.5 Few bone-spicules in periphery, cystic macular edema 21.9 0 0% 0%
The control group had visual acuity of 1.0 or better and the KVF score ranged from 83 to 100 (mean score 93). Under dark-adapted conditions, some controls could see light that was as dim as −5 log cd/m2 (dark-adapted light perceptual threshold, mean ± SD −4.4 ± 0.48 log cd/m2, see Table 2). The color blue was not identified until the stimulus light was almost 1.5 log-units higher than that to perceive light (blue color perceptual threshold −3.0 ± 0.72 log cd/m2). The NR2E3 patients needed, on average, 2.0 log-units brighter light to first perceive the light stimulus, but individual values were highly variable, ranging from −4.5 to 2.6 log cd/m2 (−2.5 ± 2.2 log cd/m2). For 4 patients, their visual perceptual threshold was the same intensity as color perception threshold. 
Table 2. 
 
Visual Threshold of Patients with NR2E3 Gene Mutation
Table 2. 
 
Visual Threshold of Patients with NR2E3 Gene Mutation
Patient (by Rank) Age (y) Dark-Adapted Light Perceptual Threshold (log cd/m2) Dark-Adapted Blue Perceptual Threshold (log cd/m2) Dark-Adapted Red Perceptual Threshold (log cd/m2) Light-Adapted Light Perceptual Threshold (log cd/m2)
P1 13 −4 −3 −3.5 0
P2 21 −4.5 −4.5 −3 −0.5
P3 28 −3.5 −2.5 −2.5 −0.5
P4 16 −3.5 −3 −3.5 −1
P5 47 −4 −2.5 −2.5 −0.5
P6 42 −1 −1 −2.5 −0.5
P7 40 −2.5 −2.5 −1.5 0
P8 60 −2 −1.5 −2 −1
P9 44 2.6 2.6
Patients (n = 9) Mean age = 35 (range 13–60) −2.5 ± 2.21 (mean ± SD) −2 ± 1.98 −2.4 ± 1.12 −0.5 ± 0.38
Controls (n = 12) Mean age = 42 (range 12–57) −4.4 ± 0.48 (mean ± SD) −3 ± 0.72 −2.8 ± 0.44 −0.7 ± 0.44
In the light-adapted red-light stimulus condition, there was a continuous blue background light and the light stimulus always was a red color, so only the colored light (cone) perceptual threshold was recorded. All subjects in the control group first perceived the stimulus light at intensity of −1.0 to 0 log cd/m2. There was no difference in the red-light perceptual threshold between patients and controls who were in the light-adapted state (−0.5 vs. −0.7 log cd/m2, respectively, P = 0.39). One patient (P9) either did not ever see a red light stimulus or simply forgot to indicate when he did. 
The mean stimulus response curves for the control and patient groups are shown in Figure 1. The individual stimulus response curve for each patient is shown in Figure 2 in order of their rank of clinical disease severity. With disease progression, there was a loss of retinal sensitivity to blue light under dark adaptation, particularly at the very dim intensities (Fig. 1). The loss of sensitivity to red light under the same testing condition was less notable. There was no observable loss of sensitivity to red light under light adaptation with clinical disease (no difference in the light-adapted red-light response curves between patient and control groups). 
Figure 1. 
 
Mean pupil constriction plotted as a function of stimulus light intensity for 12 control subjects and 9 patients with NR2E3-associated adRP under 3 stimulus conditions. Solid line: the fitted curve through the mean value at each intensity. Bars: represent 1 SD. Blue and dark red lines: the pupil responses to blue and red light stimuli presented under dark adaptation. Bright red line: the pupil responses to red stimuli presented under adaptation to blue light.
Figure 1. 
 
Mean pupil constriction plotted as a function of stimulus light intensity for 12 control subjects and 9 patients with NR2E3-associated adRP under 3 stimulus conditions. Solid line: the fitted curve through the mean value at each intensity. Bars: represent 1 SD. Blue and dark red lines: the pupil responses to blue and red light stimuli presented under dark adaptation. Bright red line: the pupil responses to red stimuli presented under adaptation to blue light.
Figure 2. 
 
Stimulus response curve of 9 patients with NR2E3-associated adRP. The graph (upper left corner) shows the mean response curves of the control group (n = 12) . The graph of each patient is shown in order of clinically determined disease progression (1–9, mildest to most severe). Blue line: the dark-adapted blue-light stimulus condition. Dark red line: the dark-adapted red-light stimulus condition. Red line: the light-adapted red-light stimulus condition. Note that as clinical disease progresses, the dark-adapted blue light response curve has reduced sensitivity and converges with the dark-adapted red-light response curve.
Figure 2. 
 
Stimulus response curve of 9 patients with NR2E3-associated adRP. The graph (upper left corner) shows the mean response curves of the control group (n = 12) . The graph of each patient is shown in order of clinically determined disease progression (1–9, mildest to most severe). Blue line: the dark-adapted blue-light stimulus condition. Dark red line: the dark-adapted red-light stimulus condition. Red line: the light-adapted red-light stimulus condition. Note that as clinical disease progresses, the dark-adapted blue light response curve has reduced sensitivity and converges with the dark-adapted red-light response curve.
The loss of blue-light sensitivity with disease progression (observed in the response curves) was accompanied by an elevation in the logI50. The logI50 of the dark-adapted blue-light stimulus condition was significantly greater in the patient group (mean −1.2 log cd/m2) compared to the control group (mean −2.5 log cd/m2, P = 0.004, Fig. 3, Table 3). In addition, an increasing blue-light logI50 correlated with increasing disease rank (r 2 = 0.7, P = 0.02). There was, however, no difference in the mean logI50 of the red-light responses under dark-adapted or light-adapted conditions between controls and patients, nor was there any correlation of these values with the extent of clinical expression of disease. 
Figure 3. 
 
Distribution plot of the logI50, and the threshold intensities for 12 controls and 9 patients. The logI50 is the intensity at which a half maximal response can be obtained and plotted on the Y axis in order of disease rank for patients and by age for controls. The threshold value is the point where the regression line intersects the criterion pupil response, Y = 5.
Figure 3. 
 
Distribution plot of the logI50, and the threshold intensities for 12 controls and 9 patients. The logI50 is the intensity at which a half maximal response can be obtained and plotted on the Y axis in order of disease rank for patients and by age for controls. The threshold value is the point where the regression line intersects the criterion pupil response, Y = 5.
Table 3. 
 
logI50 and Difference in logI50 (Dark-Adapted Blue-Light Condition Minus Dark-Adapted Red-Light Condition) of Patients with NR2E3 Gene Mutation
Table 3. 
 
logI50 and Difference in logI50 (Dark-Adapted Blue-Light Condition Minus Dark-Adapted Red-Light Condition) of Patients with NR2E3 Gene Mutation
Patients by Rank logI50
Dark-Adapted Blue-Light Stimulus Condition Dark-Adapted Red-Light Stimulus Condition Dark-Adapted Blue-Light Stimulus Condition Minus Red-Light Stimulus Condition Light Adapted Red-Light Stimulus Condition
P1 −3.04 −1.79 1.25 −0.06
P2 −1.62 −1.14 0.48 1.29
P3 −2.41 −2.02 0.39 0.17
P4 −0.71 −0.88 0 −0.06
P5 −1.16 −1.09 0.07 0.82
P6 −0.74 −0.59 0.15 −0.36
P7 −1.65 −1.56 0.09 0.07
P8 −0.56 −0.82 0 0.56
P9 0.91 −0.85 0 1.00
Patients mean ± SD (n = 9) −1.22 ± 1.15 −1.19 ± 0.49 0.27 ± 0.41 0.38 ± 0.56
Controls mean ± SD (n = 12) −2.48 ± 0.51 −1.27 ± 0.29 1.21 ± 0.40 0.60 ± 0.38
The relationship between working rods and cones under dark adaptation was assessed from retinal sensitivity to blue and red light, and expressed as a difference score (dark-adapted blue logI50 − dark-adapted red logI50). This sensitivity difference score correlated inversely with increasing disease rank (r 2 = 0.7, P = 0.02). When the dark-adapted blue and red response curves converged, and the blue-red sensitivity difference score was zero, it was presumed that there was no rod contribution and pupil responses to red light were derived from residual cone activity. 
The rod threshold, as determined from pupil responses, ranged from −5.3 to −4.0 log cd/m2 for controls (−4.7 ± 0.4 log cd/m2) and from −4.4 to −1.7 log cd/m2 for NR2E3 patients (−3.4 ± 1.1 log cd/m2), and the difference between these 2 groups was significant (P = 0.006) (Fig. 4). The rod threshold showed a general tendency to increase with disease rank but did not quite reach statistical significance (r 2 = 0.3, P > 0.05). When examining the individual values, we noted that 5 patients had a rod threshold that was within the range of control values (Table 4). Three of these patients had mild expression of clinical disease (rank 1–3). The other two patients (patients 6, 7) had the shallowest line slopes with relatively poor regression fit of their linear regression analysis, compared to the other patients (Table 4), and this may have spuriously lowered their rod threshold value. The pupil responses in the light-adapted red-light stimulus condition did not differ between controls and patients, either in the shape of the stimulus response curve or in the distribution of cone threshold values (mean cone threshold −0.7 ± 0.4 vs. −0.7 ± 0.5 log cd/m2 for controls and patients, respectively, P = 0.97; Figs. 2, 4; Table 3). 
Figure 4. 
 
The threshold intensity to evoke a criterion pupil response in 12 controls and 9 NR2E3 patients. The dark-adapted blue-light and light-adapted red-light stimulus conditions are used to define rod and cone thresholds, respectively. Note that only the pupil responses to blue light between −6 and −1 log cd/m2 are used to determine a rod threshold intensity. The pupil responses to higher intensity red lights under conditions of blue light adaptation are used to obtain the cone threshold intensity. A regression line is fitted and then extrapolated to intersect Y = 5.
Figure 4. 
 
The threshold intensity to evoke a criterion pupil response in 12 controls and 9 NR2E3 patients. The dark-adapted blue-light and light-adapted red-light stimulus conditions are used to define rod and cone thresholds, respectively. Note that only the pupil responses to blue light between −6 and −1 log cd/m2 are used to determine a rod threshold intensity. The pupil responses to higher intensity red lights under conditions of blue light adaptation are used to obtain the cone threshold intensity. A regression line is fitted and then extrapolated to intersect Y = 5.
Table 4. 
 
Pupil Threshold for Patients with NR2E3 Gene Mutation and Control Subjects
Table 4. 
 
Pupil Threshold for Patients with NR2E3 Gene Mutation and Control Subjects
Patient by Rank Dark-Adapted Blue-Light Stimulus Condition Dark-Adapted Red-Light Stimulus Condition Light-Adapted Red-Light Stimulus Condition
Threshold (log cd/m2) Slope Fit (r 2) Threshold (log cd/m2) Slope Fit (r 2) Threshold (log cd/m2) Slope Fit (r 2)
P1 −4.2 13.7 0.78 −3.8 7.5 0.86 −1.2 7.2 0.60
P2 −4.1 9.5 0.95 −2.7 8.3 0.92 −0.3 8.2 0.90
P3 −4.0 13.1 0.99 −3.9 7.8 0.83 −1.0 12 0.82
P4 −2.7 5.7 0.90 −1.6 8.2 0.90 −1.2 8.4 0.88
P5 −3.2 8.2 0.92 −2.7 7.1 0.97 0.1 9.2 0.88
P6 −4.3 4.8 0.76 −2.6 7.3 0.95 −1.4 8.0 0.75
P7 −4.4 6.7 0.86 −3.4 6.5 0.93 −0.7 8.1 0.80
P8 −1.7 13.1 0.93 −2.1 9.0 0.97 −0.4 10.3 0.93
P9 −1.9 8.2 0.87 −1.8 6.6 0.91 −0.1 9.4 0.70
Patients mean ± SD (n = 9) −3.4 ± 1.1 9.2 0.89 −2.7 ± 0.8 7.6 0.92 −0.7 ± 0.5 9.0 0.80
Controls mean ± SD (n = 12) −4.7 ± 0.4 9.9 0.96 −2.9 ± 0.5 9.5 0.95 −0.7 ± 0.4 10.2 0.87
The mean post-illumination pupil response (a difference value in the relative pupil size after light termination of a red versus blue light) was 33 for the control group compared to 28 for the NR2E3 patients. This difference was statistically significant (P = 0.02). To examine the possibility that rods contribute to this pupillographic measure of melanopsin activity, the rod threshold was correlated against the post-illumination response but this correlation was not significant (P > 0.05). 
Discussion
In these two families with adRP due to a missense mutation (p.Gly56Arg) of the NR2E3 gene, the clinical phenotype was a progressive rod-cone dystrophy. No clinical differences were noted between the two families. No patient had electrophysiologic evidence of increased sensitivity of S cones. 18,19 Increasing patient age correlated significantly with increasing severity of clinical expression. 
Under the testing condition of dark adaptation, the dimmest blue light stimuli were intended to favor rod activation, and this was supported by our finding that all control subjects reported that the first 2 or 3 perceived light stimuli had no color. The stimulus intensity for color perception was, on average, nearly 1.5 log-units above that for light perception for the control group. The patients, however, could identify a blue color at the first perceived stimulus, suggesting that some degree of cone recruitment already was present in NR2E3 patients under these same testing conditions. 
Objectively, we found that the threshold intensity for a rod-dependent pupil response (rod threshold) was elevated significantly in patient group compared to controls (−3.4 log cd/m2 vs. −4.7 log cd/m2). The logI50 of the stimulus response curve of the dark-adapted blue-light stimulus condition also was elevated significantly for the patients compared to controls and it correlated significantly with the extent of disease. This was not the case for the logI50 of the stimulus response curve of the red-light stimulus condition, either under dark adaptation or light-adaptation. 
Surprisingly, the cone threshold and pupil responses to red stimulus lights in the light-adapted state were almost identical between patients and controls. We interpreted this to indicate that either the patients retained a high degree of viable cone function or there was a relative lack of sensitivity of pupillometry, compared to ERG, for detecting early stages of cone dysfunction. 
At low-to-medium intensities, we expect pupil responses to blue light to be primarily rod generated, and responses to red light to be generated dominantly from cones but with some rod contribution. Therefore, we believe that the diminishing separation of the dark-adapted blue and red curves is due primarily to progressive rod dropout. When the two curves become superimposed in later stages of disease, then cones account for remaining pupil responses. We found that the functional relationship of rods and cones for a given subject was assessed best by the difference score of intensities generating a half-maximal response (dark-adapted logI50; difference logI50 blue − red). This measure was reduced abnormally in all patients except P1 who had the mildest clinical expression of disease and normal visual fields (Table 3). 
Rather unexpectedly, the post-illumination pupil response (the amount the pupil stays constricted after light offset) was reduced significantly in NR2E3 patients. The post-illumination pupil response following a blue light generally is melanopsin-dependent and reflective of the intrinsic photo response. We considered the possibility that the reduced post-illumination pupil response was influenced by rod loss in the patients, but the absence of correlation between the rod threshold and the post-illumination response did not support this hypothesis, nor was it due to nonspecific, external influences, such as fatigue or mechanical limitations of pupillary contraction, as the pupil response to an equivalent red light was used as the control for the non–melanopsin-mediated pupil response. It is beyond the scope of this methodology to specify the cause of this seemingly decreased intrinsic melanopsin activity, and we can only speculate that it may reflect a secondary effect of outer retinal degeneration on inner retinal melanopsin expression. 20,21 Alternatively, the reduced melanopsin activity may be due to actual loss of retinal ganglion cells in patients with NR2E3 mutation, but further studies will be needed to address this. 
In conclusion, we used chromatic pupillometry to assess objectively and characterize photoreceptor function in NR2E3 adRP patients (Gly56Arg mutation) at various stages of disease. The subjective visual response and the pupil responses to blue-light stimuli presented under dark adaptation were reduced significantly in patients compared to controls. Their pupil responses to blue light tended to diminish progressively with age and extent of clinical disease. The decreasing functional capacity of the rods was observed as an elevation of the rod threshold intensity and an increase of the light intensity required to generate a half-maximal pupil response (logI50). The relatively greater loss of rods compared to cones was demonstrated by a progressive reduction of the difference between the dark-adapted blue and red logI50
Our study demonstrated how one might analyze and use chromatic pupil responses to follow the functional status of the outer retina in an individual patient. The greater sensitivity of pupillometry permits detection and quantification of lower levels of outer photoreceptor activity compared to standard ERG. Pupillometry, thus, permits objective monitoring over a longer period of disease progression, including recovery of function, if treatment is instituted. Further studies may be directed to identifying characteristics of chromatic pupil responses that are specific or suggestive of a given gene mutation, as this might guide gene testing of patients diagnosed with retinitis pigmentosa. 
References
Boon CJ den Hollander AI Hoyong CB Cremers FP Klevering BJ Keunen JE. The spectrum of retinal dystrophies caused by mutations in the peripherin/RDS gene. Prog Retin Eye Res . 2008;27:213–235. [CrossRef] [PubMed]
Hartong DT Berson EL Dryja TP. Retinitis pigmentosa. Lancet . 2006;368:1795–1809. [CrossRef] [PubMed]
Sullivan LS Browne SJ Birch DG Prevalence of disease-causing mutations in families with autosomal dominant retinitis pigmentosa: a screen of known genes in 200 families. Invest Ophthalmol Vis Sci . 2006;47:3052–3064. [CrossRef] [PubMed]
Gire AI Sullivan LS Bowne SJ The Gly56Arg mutation in NR2E3 accounts for 1–2% of autosomal dominant retinitis pigmentosa. Mol Vis . 2007;13:1970–1975. [PubMed]
Coppieters F Leroy BP Beyson D Recurrent mutation in the first zinc finger of the orphan nuclear receptor NR2E3 causes autosomal dominant retinitis pigmentosa. Am J Hum Genet . 2007;81:147–157. [CrossRef] [PubMed]
Jacobson SG Aleman TS Cideciyan AV Defining the residual vision in Leber congenital amaurosis caused by RPE65 mutation. Invest Ophthalmol Vis Sci . 2009;50:2368–2375. [CrossRef] [PubMed]
Lupo S Grenga PL Vingolo EM. Fourier-domain optical coherence tomography and microperimetry findings in retinitis pigmentosa. Am J Ophthalmol . 2011;151:106–111. [CrossRef] [PubMed]
Loewenfeld IE. The Pupil . Detroit, MI: Wayne State Press; 1993:84–274.
Alexandridis E Weddigen A. Pupillary light reflexes in Herododegeneratio pigmentosa retinae. Albrecht Von Graefes Arch Klin Exp Ophthalmol . 1971;182:250–260. [CrossRef] [PubMed]
Birch EE Birch DG. Pupillometric measures of retinal sensitivity in infants and adults with retinitis pigmentosa. Vision Res . 1987;27:499–505. [CrossRef] [PubMed]
Kardon R Anderson SC Damarjian TG Grace EM Stone E Kawasaki A. Chromatic pupillometry in patients with retinitis pigmentosa. Ophthalmology . 2011;118:376–381. [CrossRef] [PubMed]
Park JC Moura AL Raza AS Rhee DW Kardon RH Hood DC. Toward a clinical protocol for assessing rod, cone, and melanopsin contributions to the human pupil response. Invest Ophthalmol Vis Sci . 2011;52:6624–6635. [CrossRef] [PubMed]
Jacobson SG Cideciyan AV Aleman TS Human retinal disease from AIPL1 gene mutations: foveal cone loss with minimal macular photoreceptors and rod function remaining. Invest Ophthalmol Vis Sci . 2011;52:70–79. [CrossRef] [PubMed]
Kawasaki A Munier FL Léon L Kardon RH. Pupillometric quantification of residual rod and cone activity in Leber congenital amaurosis. Arch Ophthalmol . 2012;130:798–800. [CrossRef] [PubMed]
Kardon RH Anderson SC Damarjian TG Grace EM Stone E Kawasaki A. Chromatic pupil responses: preferential activation of the melanopsin-mediated versus outer photoreceptor-mediated pupil light reflex. Ophthalmology . 2009;116:1564–1573. [CrossRef] [PubMed]
Esterman B. Grid for scoring visual fields: I. Tangent screen. Arch Ophthalmol . 1967;77:780–786. [CrossRef] [PubMed]
Esterman B. Grid for scoring visual fields: II. Perimeter. Arch Ophthalmol . 1968;79:400–406. [CrossRef] [PubMed]
Milam AH Jacobson SG. Photoreceptor rosettes with blue cone opsin immunoreactivity in retinitis pigmentosa. Ophthalmology . 1990;97:1620–1631. [CrossRef] [PubMed]
Roman AJ Jacobson SG. S cone-driven but not S cone-type electroretinograms in the enhanced S cone syndrome. Exp Eye Res . 1991;53:685–690. [CrossRef] [PubMed]
Ruggiero L Allen CN Brown RL Robinson DW. The development of melanopsin-containing retinal ganglion cells in mice with early retinal degeneration. Eur J Neurosci . 2009;29:359–367. [CrossRef] [PubMed]
Wan J Zheng H Hu BY Acute photoreceptor degeneration down-regulates melanopsin expression in adult rat retina. Neurosci Lett . 2006;400:48–52. [CrossRef] [PubMed]
Footnotes
 Supported in part by a grant from the Foundation for Research in Ophthalmology and Loterie Romande Switzerland (AK), the Department of Veterans Affairs Rehabilitation Research and Development Division, Department of Defense and the Pomerantz Chair in Ophthalmology (RK), the Research to Prevent Blindness (New York, New York), and the French Ministry of Health (PHRC #2008-A01238-47), the Foundations RETINA France, and UNADEV (CH).
Footnotes
 Disclosure: A. Kawasaki, None; S.V. Crippa, None; R. Kardon, None; L. Leon, None; C. Hamel, None
Figure 1. 
 
Mean pupil constriction plotted as a function of stimulus light intensity for 12 control subjects and 9 patients with NR2E3-associated adRP under 3 stimulus conditions. Solid line: the fitted curve through the mean value at each intensity. Bars: represent 1 SD. Blue and dark red lines: the pupil responses to blue and red light stimuli presented under dark adaptation. Bright red line: the pupil responses to red stimuli presented under adaptation to blue light.
Figure 1. 
 
Mean pupil constriction plotted as a function of stimulus light intensity for 12 control subjects and 9 patients with NR2E3-associated adRP under 3 stimulus conditions. Solid line: the fitted curve through the mean value at each intensity. Bars: represent 1 SD. Blue and dark red lines: the pupil responses to blue and red light stimuli presented under dark adaptation. Bright red line: the pupil responses to red stimuli presented under adaptation to blue light.
Figure 2. 
 
Stimulus response curve of 9 patients with NR2E3-associated adRP. The graph (upper left corner) shows the mean response curves of the control group (n = 12) . The graph of each patient is shown in order of clinically determined disease progression (1–9, mildest to most severe). Blue line: the dark-adapted blue-light stimulus condition. Dark red line: the dark-adapted red-light stimulus condition. Red line: the light-adapted red-light stimulus condition. Note that as clinical disease progresses, the dark-adapted blue light response curve has reduced sensitivity and converges with the dark-adapted red-light response curve.
Figure 2. 
 
Stimulus response curve of 9 patients with NR2E3-associated adRP. The graph (upper left corner) shows the mean response curves of the control group (n = 12) . The graph of each patient is shown in order of clinically determined disease progression (1–9, mildest to most severe). Blue line: the dark-adapted blue-light stimulus condition. Dark red line: the dark-adapted red-light stimulus condition. Red line: the light-adapted red-light stimulus condition. Note that as clinical disease progresses, the dark-adapted blue light response curve has reduced sensitivity and converges with the dark-adapted red-light response curve.
Figure 3. 
 
Distribution plot of the logI50, and the threshold intensities for 12 controls and 9 patients. The logI50 is the intensity at which a half maximal response can be obtained and plotted on the Y axis in order of disease rank for patients and by age for controls. The threshold value is the point where the regression line intersects the criterion pupil response, Y = 5.
Figure 3. 
 
Distribution plot of the logI50, and the threshold intensities for 12 controls and 9 patients. The logI50 is the intensity at which a half maximal response can be obtained and plotted on the Y axis in order of disease rank for patients and by age for controls. The threshold value is the point where the regression line intersects the criterion pupil response, Y = 5.
Figure 4. 
 
The threshold intensity to evoke a criterion pupil response in 12 controls and 9 NR2E3 patients. The dark-adapted blue-light and light-adapted red-light stimulus conditions are used to define rod and cone thresholds, respectively. Note that only the pupil responses to blue light between −6 and −1 log cd/m2 are used to determine a rod threshold intensity. The pupil responses to higher intensity red lights under conditions of blue light adaptation are used to obtain the cone threshold intensity. A regression line is fitted and then extrapolated to intersect Y = 5.
Figure 4. 
 
The threshold intensity to evoke a criterion pupil response in 12 controls and 9 NR2E3 patients. The dark-adapted blue-light and light-adapted red-light stimulus conditions are used to define rod and cone thresholds, respectively. Note that only the pupil responses to blue light between −6 and −1 log cd/m2 are used to determine a rod threshold intensity. The pupil responses to higher intensity red lights under conditions of blue light adaptation are used to obtain the cone threshold intensity. A regression line is fitted and then extrapolated to intersect Y = 5.
Table 1. 
 
Clinical Features of Nine Patients with NR2E3 Gene Mutation (p.Gly56Arg)
Table 1. 
 
Clinical Features of Nine Patients with NR2E3 Gene Mutation (p.Gly56Arg)
Patient by Rank and Family (A or B) Age (y) / Sex (M/F) Symptoms VA Fundus CVF Defect (MD in dB) KVF Score Scotopic ERG (% of Normal −25 dB B-Wave Amplitude) Photopic ERG (% of Normal 0 dB B-Wave Amplitude )
P1 (B) 13/M None 1.0 Few atrophic areas in mid periphery, pigment clump inferotemporal periphery −0.9 100 27% 78%
P2 (A) 21/M None 1.0 Peripapillary depigmentation −0.1 87 30% 45%
P3 (B) 28/M Night blindness 0.7 Normal fundus 14.6 92 Not done Not done
P4 (B) 16/F Night blindness 1.0 Pigment atrophy in peripapillary and mid peripheral areas 13.4 51 0% 70%
P5 (A) 47/M Night blindness, photophobia 0.5 Atrophic perifoveal ring, pigment clumps in temporal periphery 13.1 70 0% 55%
P6 (B) 42/F Night blindness, photophobia 0.7 White deposit along vascular arcades, few pigment clumps in mid periphery 16.5 25 26% 54%
P7 (A) 40/F Night blindness 1.0 Normal fundus 0 28 0% 70%
P8 (B) 60/M Night blindness 0.5 Many bone-spicules in the mid periphery, pigment clumps in extreme periphery, attenuated vessels 18.3 0 0% 0%
P9 (A) 44/M Night blindness, mild photophobia 0.5 Few bone-spicules in periphery, cystic macular edema 21.9 0 0% 0%
Table 2. 
 
Visual Threshold of Patients with NR2E3 Gene Mutation
Table 2. 
 
Visual Threshold of Patients with NR2E3 Gene Mutation
Patient (by Rank) Age (y) Dark-Adapted Light Perceptual Threshold (log cd/m2) Dark-Adapted Blue Perceptual Threshold (log cd/m2) Dark-Adapted Red Perceptual Threshold (log cd/m2) Light-Adapted Light Perceptual Threshold (log cd/m2)
P1 13 −4 −3 −3.5 0
P2 21 −4.5 −4.5 −3 −0.5
P3 28 −3.5 −2.5 −2.5 −0.5
P4 16 −3.5 −3 −3.5 −1
P5 47 −4 −2.5 −2.5 −0.5
P6 42 −1 −1 −2.5 −0.5
P7 40 −2.5 −2.5 −1.5 0
P8 60 −2 −1.5 −2 −1
P9 44 2.6 2.6
Patients (n = 9) Mean age = 35 (range 13–60) −2.5 ± 2.21 (mean ± SD) −2 ± 1.98 −2.4 ± 1.12 −0.5 ± 0.38
Controls (n = 12) Mean age = 42 (range 12–57) −4.4 ± 0.48 (mean ± SD) −3 ± 0.72 −2.8 ± 0.44 −0.7 ± 0.44
Table 3. 
 
logI50 and Difference in logI50 (Dark-Adapted Blue-Light Condition Minus Dark-Adapted Red-Light Condition) of Patients with NR2E3 Gene Mutation
Table 3. 
 
logI50 and Difference in logI50 (Dark-Adapted Blue-Light Condition Minus Dark-Adapted Red-Light Condition) of Patients with NR2E3 Gene Mutation
Patients by Rank logI50
Dark-Adapted Blue-Light Stimulus Condition Dark-Adapted Red-Light Stimulus Condition Dark-Adapted Blue-Light Stimulus Condition Minus Red-Light Stimulus Condition Light Adapted Red-Light Stimulus Condition
P1 −3.04 −1.79 1.25 −0.06
P2 −1.62 −1.14 0.48 1.29
P3 −2.41 −2.02 0.39 0.17
P4 −0.71 −0.88 0 −0.06
P5 −1.16 −1.09 0.07 0.82
P6 −0.74 −0.59 0.15 −0.36
P7 −1.65 −1.56 0.09 0.07
P8 −0.56 −0.82 0 0.56
P9 0.91 −0.85 0 1.00
Patients mean ± SD (n = 9) −1.22 ± 1.15 −1.19 ± 0.49 0.27 ± 0.41 0.38 ± 0.56
Controls mean ± SD (n = 12) −2.48 ± 0.51 −1.27 ± 0.29 1.21 ± 0.40 0.60 ± 0.38
Table 4. 
 
Pupil Threshold for Patients with NR2E3 Gene Mutation and Control Subjects
Table 4. 
 
Pupil Threshold for Patients with NR2E3 Gene Mutation and Control Subjects
Patient by Rank Dark-Adapted Blue-Light Stimulus Condition Dark-Adapted Red-Light Stimulus Condition Light-Adapted Red-Light Stimulus Condition
Threshold (log cd/m2) Slope Fit (r 2) Threshold (log cd/m2) Slope Fit (r 2) Threshold (log cd/m2) Slope Fit (r 2)
P1 −4.2 13.7 0.78 −3.8 7.5 0.86 −1.2 7.2 0.60
P2 −4.1 9.5 0.95 −2.7 8.3 0.92 −0.3 8.2 0.90
P3 −4.0 13.1 0.99 −3.9 7.8 0.83 −1.0 12 0.82
P4 −2.7 5.7 0.90 −1.6 8.2 0.90 −1.2 8.4 0.88
P5 −3.2 8.2 0.92 −2.7 7.1 0.97 0.1 9.2 0.88
P6 −4.3 4.8 0.76 −2.6 7.3 0.95 −1.4 8.0 0.75
P7 −4.4 6.7 0.86 −3.4 6.5 0.93 −0.7 8.1 0.80
P8 −1.7 13.1 0.93 −2.1 9.0 0.97 −0.4 10.3 0.93
P9 −1.9 8.2 0.87 −1.8 6.6 0.91 −0.1 9.4 0.70
Patients mean ± SD (n = 9) −3.4 ± 1.1 9.2 0.89 −2.7 ± 0.8 7.6 0.92 −0.7 ± 0.5 9.0 0.80
Controls mean ± SD (n = 12) −4.7 ± 0.4 9.9 0.96 −2.9 ± 0.5 9.5 0.95 −0.7 ± 0.4 10.2 0.87
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