Development of a Chromatic Pupillography Protocol for the First Gene Therapy Trial in Patients With CNGA3-Linked Achromatopsia

Abstract

Citation: Lisowska J, Lisowski L, Kelbsch C, et al. Development of a chromatic pupillography protocol for the first gene therapy trial in patients with CNGA3-linked achromatopsia. Invest Ophthalmol Vis Sci. 2017;58:127458: -128258: . DOI:10.1167 PURPOSE. To establish a feasible and sensitive pupillographic protocol to assess outer and inner retinal function for the first gene therapy trial in achromatopsia patients (ACHM) with mutations in CNGA3.METHODS. Twenty-seven CNGA3-ACHM patients and 22 age-matched control subjects were tested using chromatic pupillography. Three different protocols were established to assess the pupillary light reflex parameters and to create the final protocol. In the individual protocols, various stimulus parameters (i.e., intensity, duration, wavelength, adaptation states) were applied to evaluate the impact of these stimuli on the pupillary response in untreated ACHM patients.RESULTS. In the light-adapted conditions, CNGA3-ACHM patients showed significantly reduced maximal amplitudes compared with the control group when using a 1-second high intensity (28-lux corneal illumination) blue or red stimulus (P < 0.005). In the dark-adapted conditions, CNGA3-ACHM patients unexpectedly revealed significantly increased maximal amplitudes when stimulating with red (1 second) or blue (4 ms and 1 second) stimuli of low intensity (0.01-lux corneal illumination; P < 0.05). Pupil responses of CNGA3-ACHM patients after high intensity (28 lux) red and blue 1-second stimuli were within the normal range.CONCLUSIONS. Chromatic pupillography demonstrated significant reduced pupil responses to stimuli addressing primarily cone function, an increased sensitivity to rod-favoring stimuli and evidence for disinhibition of intrinsically photosensitive retinal ganglion cells in CNGA3-ACHM patients. A final protocol was established based on these findings. These conclusions may be useful for the objective assessment of efficacy gained by gene therapy or other innovative interventions in this hereditary retinal disorder.Keywords: chromatic pupillography, achromatopsia, CNGA3, ipRGC A chromatopsia (ACHM) is an inherited autosomal recessive congenital retinal disease, with a prevalence of one in 30,000.1 The most characteristic symptom is the inability to discriminate colors due to the loss of cone photoreceptor function. Achromatopsia patients also suffer from severely reduced visual acuity, nystagmus, and photophobia. 2 Rarely, individuals have incomplete ACHM, in which one or more cone types may be partially functioning. 3 The clinical findings of incomplete ACHM are similar to the complete form, but milder. Fundus examination is usually normal, and electrophysiological testing demonstrates absent cone responses and normal or near-normal rod function. [2][3][4][5] To date, mutations in six genes have been shown to be associated with ACHM: CNGA3 and CNGB3 encode the a and b subunits of the cGMP-gated cation channel, 6 GNAT2 the a subunit of the cone-specific G-protein transducing, 7 PDE6C and PDE6H code for the ...

Standards in Pupillography

“…For a reliable interpretation of the data and to facilitate replication of findings, the absolute pupil baseline diameter before the stimulation should be reported. This metric varies widely across participants with a characteristic decreasing pupil size with age (2) and hints for smaller pupil sizes in specific retinal diseases, e.g., in CNGA3-linked Achromatopsia (35). Further analyses should usually be based on relative values as pupillary responses are dependent on the initial baseline diameter, which should be obtained during a sufficiently long recording period to ensure a steady and reliable estimate.…”

“…There are examples of chromatic pupillometry methodologies that provide initial efforts to separate rod and cone function through the careful control of the wavelength, irradiance, size and duration of the test stimuli; the degree of separation of rod and cone (and melanopsin) function that these conditions provide is still to be determined. At light levels below cone threshold, short wavelength lights are presented in the dark to bias the response to rods, with the PIPR amplitudes minimized under such conditions (9, 11, 35). To ensure maximal rod sensitivity, the pre-stimulus dark adaptation time should be at least 30 min (91); although this is not practical for all clinical protocols, shorter periods will influence the relative rod and cone sensitivity to the test stimuli.…”

“…On the other hand, there are pupillographic clinical studies in humans in order to better understand the pupil circuitry [e.g., (17, 18)] and the pathomechanism and remaining retinal functionality of certain diseases, e.g., glaucoma (19–23), Retinitis pigmentosa (9, 24–27), age-related macular degeneration (28–30), diabetes (31–33) or hereditary optic neuropathy (34). Furthermore, pupillography comes into use in clinical observational and therapy studies like gene therapy of hereditary retinal degenerations (35), studies on attention-modulation (36), in chronobiology [(37–39), for a review see (40)] and in psychopathology, psychiatric disorders and neurodegenerative conditions (41–45). Additionally, pupillography is indispensable in sleepiness-related research and the pupillographic sleepiness test (PST) has been developed into an objective measures of day time sleepiness (46, 47).…”

Effect of Stimulus Intensity and Visual Field Location on Rod- and Cone-Mediated Pupil Response to Focal Light Stimuli

“…Indeed, these and subsequent studies showed that the PLR for chromatic light may potentially be used for diagnosis and monitoring disease progression as well as determination of treatment efficacy and safety in retinal and optic nerve diseases. 5,[7][8][9][10][13][14][15][16] Recently, Park et al 17 characterized the PLR mediated by the different photoreceptor classes when using smaller stimulus sizes presented in the center of the visual field (VF) over a large range of light intensities. Chibel et al 18 provided evidence that the function of rods and cones at different locations in the central (16.28) VF can be assessed using smaller (Goldmann size III, 0.438) red and blue light stimuli at 1000 and 200 cd/m 2 , respectively, under mesopic (0.05 cd/m 2 uniform white light) adaptation.…”

Cone-Mediated Function Correlates to Altered Foveal Morphology in Preterm-Born Children at School Age

“…Stimulus projection was achieved with a time-triggered Espion Ganzfeld ColorDome Stimulator (Espion E 2 ; Diagnosys LLC, Lowell, MA, USA). Based on the protocols described by Park et al, 23 Kardon et al, 24 Kawasaki et al, 25 and Lisowska et al, 26 blue and red stimuli were displayed with a duration of 1 second under darkand light-adapted conditions. All preterm-born and term-born children were tested with a protocol containing three repeats of fixed illuminances (0.01 cd/m 2 blue stimuli at a wavelength of 460 nm and 100 cd/m 2 red stimuli at a wavelength of 640 nm).…”