RDH12 Mutations Cause a Severe Retinal Degeneration with Relatively Spared Rod Function

Retina RDH12 Mutations Cause a Severe Retinal Degeneration With Relatively Spared Rod Function

Tomas S. Aleman,1,2 Katherine E. Uyhazi,1 Leona W. Serrano,1 Vidyullatha Vasireddy,2 Scott J. Bowman,1 Michael J. Ammar,1 Denise J. Pearson,1 Albert M. Maguire,1,2 and Jean Bennett1,2 1Scheie Eye Institute at the Perelman Center for Advanced Medicine, Philadelphia, Pennsylvania, United States 2Department of Ophthalmology, Center for Advanced Ocular and Retinal Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States

Correspondence: Tomas S. Aleman, PURPOSE. To describe the retinal phenotype of pediatric patients with mutations in the retinol Perelman Center for Advanced Med- dehydrogenase 12 (RDH12) gene. icine, University of Pennsylvania, 3400 Civic Center Boulevard, Phila- METHODS. Twenty-one patients from 14 families (ages 2–17 years) with RDH12-associated delphia, PA 19104, USA; inherited retinal degeneration (RDH12-IRD) underwent a complete ophthalmic exam and [email protected]. imaging with spectral domain optical coherence tomography (SD-OCT) and near infrared and TSA and KEU are joint first authors. short-wavelength fundus autofluorescence. Visual field extent was measured with Goldmann kinetic perimetry, visual thresholds with dark-adapted static perimetry or with dark-adapted Submitted: May 29, 2018 chromatic full-field stimulus testing (FST) and transient pupillometry. Accepted: August 29, 2018 RESULTS. Visual acuity ranged from 20/40 to light perception. There was parafoveal Citation: Aleman TS, Uyhazi KE, Ser- rano LW, et al. RDH12 mutations depigmentation or atrophic maculopathies accompanied by midperipheral intraretinal cause a severe retinal degeneration pigment migration. SD-OCT revealed foveal thinning in all patients and detectable but with relatively spared rod function. thinned outer nuclear layer (ONL) at greater eccentricities from the fovea. Photoreceptor Invest Ophthalmol Vis Sci. outer segment (POS) signals were only detectable in small pockets within the central retina. 2018;59:5225–5236. https://doi.org/ Measurable kinetic visual fields were limited to small (<5–108) central islands of vision. 10.1167/iovs.18-24708 Electroretinograms were reported as undetectable or severely reduced in amplitude. FST sensitivities to a 467 nm stimulus were rod-mediated and reduced on average by ~2.5 log units. A thinned central ONL colocalized with severely reduced to nondetectable cone- mediated sensitivities. Pupillometry confirmed the psychophysically measured abnormalities.

CONCLUSIONS. RDH12-IRD causes an early-onset, retina-wide disease with particularly severe central retinal abnormalities associated with relatively less severe rod photoreceptor dysfunction, a pattern consistent with an early-onset cone-rod dystrophy. Severely abnormal POS but detectable ONL in the pericentral and peripapillary retina suggest these regions may become targets for gene therapy. Keywords: Leber congenital amaurosis, RDH12, optical coherence tomography, rods, cones, cone-rod dystrophy, photoreceptors

eber congenital amaurosis (LCA) and early onset retinal the inner segment against retinaldehyde-induced cytotoxici- L degenerations (EORD) describe a molecularly and clinically ty.10–12 heterogeneous group of inherited retinal degenerations (IRDs) The most frequent retinal phenotype in patients with characterized by severe vision loss recognized during the ﬁrst RDH12 mutations is an autosomal recessive inherited retinal year of life or early in infancy.1–3 The molecular cause of LCA/ degeneration (IRD) within the spectrum of severity of LCA, EORD has been elucidated in a large proportion of patients and although relatively milder phenotypes have been categorized as there are 25 causative genes identiﬁed to date (https://sph.uth. forms of EORD or juvenile-onset retinitis pigmentosa (RP). edu/retnet/disease.htm, available in the public domain).4–7 Rarely, RDH12 can segregate with a milder phenotype in an Mutations in the retinol dehydrogenase 12 gene (RDH12), autosomal dominant fashion.6 Independent of the age at which maps to a locus on chromosome 14 (14q23.3-24.1) presentation of the recessive disease, the phenotypic expres- known as LCA13, accounts for about 4% of all autosomal sion includes a progressive, retina-wide pigmentary retinopathy recessive LCA, a disease that affects about 20% of all children with peripapillary sparing and prominent central changes that that attend schools for the blind.1,4,8,9 The gene encodes a result in early visual acuity (VA) loss.13 protein member of the family of retinol dehydrogenases that RDH12-associated EORDs (RDH12-EORD) or LCA (RDH12- localizes to the photoreceptor inner segment of rods and LCA) do not appear to show the structural functional cones. RDH12 and RDH8 provide most of the reductase dissociation observed in other forms of LCA, particularly LCA activity in the inner and outer segment and reduce all-trans- associated with mutations in RPE65 (RPE65-LCA), a disease retinal released after photoactivation, a step needed for caused by loss-of-function of another visual cycle protein photopigment regeneration.10 More importantly, there is (RPE65) that has successfully been treated with gene therapy.14 evidence that by reducing free all-trans-retinal, RDH12 protects Despite potential differences in disease mechanisms and

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expression, there is reasonable hope that other genetic forms near infrared reflectance (NIR-REF) and autofluorescence (NIR- of IRD within the spectrum of EORD may be similarly treatable FAF) images were obtained during the acquisition of the OCTs with gene augmentation. To increase our understanding of the with overlapping 308 and/or 558 fields. In a subset of patients retinal structural changes and associated visual dysfunction (n ¼ 5) short-wavelength autofluorescence (SW-FAF) imaging resulting from RDH12 mutations, we studied a relatively large was performed in a similar manner. group of pediatric patients with RDH12-LCA/EORD using psychophysics, measures of the transient pupillary light reflex Pupillometry (TPLR), and multimodal retinal imaging. The ultimate goal was to determine the treatment potential and targets as well as The direct transient pupillary light reflex (TPLR) was elicited eligibility of patients for future gene augmentation clinical trials by full-field, short duration (100 msec), chromatic stimuli, for this condition. delivered using the stimulator of the system used for full field stimulus testing (FST; Espion 3; Diagnosys, Lowell, MA, USA).21 The pupil was illuminated and imaged with a head-mounted METHODS near infrared LED and infrared-sensitive video camera, respec- tively, and the images of the pupil were recorded and Patients processed by an eye-tracker (EyeFrame; Arrington Research, Scottsdale, AZ, USA). A separate program (VoltagePoint; Twenty-one patients, ages 2–17 years, representing 14 families Arrington Research) synchronized both instruments through diagnosed with RDH12-LCA and RDH12-EORD were included a signal input delivered by the software of the stimulator in this prospective, cross-sectional study. Genotyping was system.22 TPLR luminance response functions were recorded undertaken using saliva samples when not already determined. with increasing intensities of scotopically matched blue (peak Informed consent and assent were obtained after explanation 467 nm; from 4.25 to þ4.25 log scot-cd.s.m2) and red (peak of the nature of the study; procedures complied with the 637 nm; 4.25 to þ3.25 log scot-cd.s.m2) lights presented at Declaration of Helsinki and were approved by the institutional 0.5 log unit increments in the dark-adapted (>45 minutes) review board (IRB #808828, 815348). All patients underwent a state.22 The recording window was 4 seconds long. Interstim- comprehensive eye examination. Fixation pattern and tracking ulus intervals were long enough to allow the pupil to return to was documented in the youngest patient (Patient 1, P1); the its baseline diameter and varied from ~15 seconds at lower rest of the patients had VAs measured with Snellen visual acuity intensities to ~30 seconds at the higher end. In patients, the charts. Testing was done for each eye independently; for starting intensity was arbitrarily selected at ~2 log units above clarity, imaging results illustrated in figures correspond to the normal dark-adapted TPLR thresholds.22 TPLR amplitudes were eye with better acuity or favored by the patient. measured as the difference in horizontal pupil diameter between baseline and a fixed 0.6-second time after stimulus Retinal Imaging presentation; TPLR thresholds were defined as pupil contraction amplitudes reaching a 0.3 mm criterion response as Imaging was performed with spectral domain optical coher- defined previously.22 A video clip was recorded by the ence tomography (SD-OCT; Spectralis; Heidelberg Engineering, pupillometer, which was used to perform manual measures Carlsbad, CA, USA) with 9 mm-long horizontal sections in patients with wandering eye movements or large-amplitude crossing the anatomical fovea, extending when possible into nystagmus in which the automated software was unable to the midperipheral retina (n ¼ 14 patients). Our SD-OCT calculate pupil diameter. scanning protocol in young patients, analyses, and normative data have been published.15–17 In brief, segmentation of SD- OCT images was performed with the built-in segmentation Visual Psychophysics software of the Spectralis OCT system. A separate imaging Visual field extent was determined with Goldmann kinetic analysis software (http://imagej.nih.gov/ij/links.html, available perimetry. Visual sensitivity was measured in dark-adapted in the public domain) was used to generate longitudinal (>45 minutes) patients using FST.21,23,24 Mydriatics were reflectivity profiles from exported images and help supervise delivered after pupillometry and additional dark-adaptation 18–20 and correct automatic segmentation errors. ‘‘Retinal (15–20 minutes) occurred while pupils dilated. Dark-adapted thickness’’ was defined as the distance between the signal psychophysic thresholds were determined with chromatic peak at the vitreoretinal interface (the internal limiting (blue, peak 467 nm and red, peak 637 nm) stimuli using a membrane [ILM]) and the posterior boundary of the major thresholding algorithm built-in into a computer-driven electro- signal peak that corresponds to the basal retinal pigment retinography (ERG) system.23 Two to three separate determi- epithelium/Bruch’s membrane complex (RPE/BrM). In normal nations of the sensitivity were performed for each stimulus subjects, the RPE/BrM is the last reflectivity within the 4–6 type. Sensitivity differences between each of the determina- signals that are identifiable in the outer retina.20 In patients, tions or runs (run1 minus run2, run1-run3) were used to the presumed RPE signal was sometimes the only signal in the estimate the variability of the measurements. Estimates in outer retina and often merged with signals from the anterior patients (1.9 6 3.8 dB) were similar to values determined in choroid. The RPE/BrM peak intensity was then specified young normal subjects (n ¼1.6 6 4.1 dB, P ¼ 0.89) from this manually by considering the properties of the backscattering study, and were comparable to previous reports.21,23 One signal originating from layers vitread and sclerad to it.19 The patient was too variable (differences >30 dB), so her values outer nuclear layer (ONL) thickness was defined as the major were not included in the analyses. Spectral sensitivity intraretinal hyporeflective signal bracketed between the outer differences were used to determine photoreceptor mediation plexiform layer (OPL) and the external limiting membrane of each stimulus condition.21 All patients, except Patient 1 (P1) (ELM) or the BrM/RPE signal when the ELM was not and P2 were able to complete the FST test in each eye; results detectable. To avoid ambiguities in the determination of the in P5 were too variable, were considered not reliable, and were ELM in severely degenerated regions, the term ‘‘outer retinal excluded from analyses. Goldmann kinetic perimetry was thickness’’ was adopted to define the thickness between the performed in all but the youngest patient (Table). Five patients OPL and RPE/BrM. The ‘‘inner retinal thickness’’ was defined (P12–P14, P17, P20) were able to complete light-adapted as the distance between the ILM and the OPL.15,17,19 En face achromatic and dark-adapted chromatic (500 nm and 650 nm)

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Visual Kinetic Foveal Acuity‡ Refraction§ Perimetry# Thickness (lm) Age*/ Age at RDH12 Variants Mutation Pt ID Sex Diagnosis Ancestry Allele1/Allele2† Type OD OS OD OSMacula ERGs OD OS OD OS

P1 2/Fjj 2 German, Scottish, Cherokee Ala269del/Val233Leu Null/MS F/F F/F þ4.63 þ4.00 nl np np np 96 85 P2 4/Fjj 3 German, Irish, English, Dutch Ala269del/Arg295Stop Null/Null 20/125 20/125 þ1.50 þ1.50 a np 10 10 100 154 P3 6/Mjj birth German, Irish, English, Dutch Ala269del/Arg295Stop Null/Null 20/200 20/200 þ1.38 þ1.38 m nd 4 5 114 96 P4 6/Mjj 1 German Ala269del/Ala126Glu Null/MS 20/200 20/125 þ3.75 þ2.50 m np 5 7 85 77 P5 7/F 3 Greek, Italian Arg62Stop/Leu99Ile Null/MS 20/250 20/50 þ4.50 þ4.00 m np 10þT5þT 85 100 P6 7/Fjj 1 German, Scottish, Cherokee Ala269del/Val233Leu Null/MS 20/125 20/70 þ0.50 þ1.25 m np 8 8 104 131 P7 8/M 2 mo Iranian, Pakistani, Indian Gly76Arg/Ala126Val MS/MS 20/200 20/400 þ4.63 þ5.25 m nd 8 5 94 94 RDH12 P8 8/Mjj 4 German, Irish, Czech Arg295Stop/Asp101Gly Null/MS 20/80 20/50 þ1.50 þ1.50 m abn 8 8 84 97 P9 9/M 6 Welsh, Polish Val233Leu/Ser134Pro MS/MS 20/400 20/400 þ1.87 þ2.00 m np 9 5 129 157

P10 9/F 1 Scottish lIe22Gly/Leu214Pro MS/MS 20/300 20/400 þ4.50 þ5.25 p-coloboma np 3þT5þT9683 -LCA P11 10/F 2 German Ala269del/Ala269del Null/Null 20/70 20/60 þ2.50 þ3.25 m nd 10 3 90 96 P12 10/F 2 Mexican Arg295Stop/Arg295Stop Null/Null 20/100 20/60 þ2.25 þ3.25 m np 10 10 108 112 P13 11/F birth German, Irish, English, Italian Ala269del/Thr49Met Null/MS 20/60 20/125 þ2.25 þ3.25 p-coloboma abn 2 2 76 34 P14 11/Fjj 3 German, Irish, Czech Arg295Stop/Asp101Gly Null/MS 20/60 20/40 þ2.50 þ1.50 m abn 6 7 77 111 P15 11/Mjj birth German, Irish, Scottish Ala269del/Ala126Glu Null/MS 20/70 20/100 þ3.37 þ3.25 m np 7þT3þT8078 P16 11/Fjj 1 Irish, Scottish, Welsh Ala269del/Arg295Stop Null/Null 20/125 20/125 þ3.00 þ3.50 m nd 8 9 130 133 P17 11/Fjj 2 German, Scottish, Cherokee Ala269del/Val233Leu Null/MS 20/250 20/125 þ1.87 þ1.25 m nd 4 3 87 57 P18 13/F 1 German, Irish, Polish Ala269del/Thr45Ala Null/MS 20/400 20/400 þ3.37 þ4.75 m np 5 9 83 84 P19 13/Mjj 1 Irish, Scottish, Welsh Ala269del/Arg295Stop Null/Null 20/60 20/300 þ2.37 þ2.87 p-coloboma nd 9þT2þT 125 91 P20 13/Fjj 3 German, Scottish, Cherokee Ala269del/Val233Leu Null/MS 20/100 20/300 þ4.87 þ3.75 m na 10 10 112 85 P21 17/F 3 Lebanese Val238ins/Val238ins Null/Null LP 20/800 5.12 2.62 p-coloboma np 2 2 12 37 np, not performed; na, not available; nl, normal; abn., severely reduced ERG amplitudes; nd, nondetectable; a, well-delimited chorioretinal atrophy; m, maculopathy; p-coloboma, bilateral pseudo- coloboma. * Age in years. † Predicted amino acid change; MS, missense mutation. ‡ FF, ﬁxates and follows; LP, light perception. § Spherical equivalent. jj Patients are siblings: P1, P6, P17, & P20; P2 & P3; P4 & P15; P8 & P14; P16 & P19. All families originate from the United States; closest ancestral origin tabulated. IOVS # Extent in eccentricity to the largest extent of the central isopter (Goldmann V-4e target); þT denotes small temporal island of vision separated from the central residual island. j coe 2018 October j o.59 Vol. j o 12 No. j 5227 Retinal Structure and Function in RDH12-LCA IOVS j October 2018 j Vol. 59 j No. 12 j 5228

automated static perimetry using 200 ms duration, 1.78 diameter stimuli, in a modiﬁed Humphrey Field Analyzer (HFA II-i; Carl Zeiss Meditec, Dublin, CA, USA), following published methodology.25–27 Thresholds were measured along the horizontal meridian at 28 intervals, extending to 308 of eccentricity, corresponding to the retinal region scanned with SD-OCT.27 Photoreceptor mediation was assessed at each locus using the sensitivity difference between the two colors.25

RESULTS Twenty-one pediatric patients from 14 families carrying homozygous or compound heterozygous mutations in RDH12 were included in this study (Table). These included frameshift mutations leading to premature stop codons, null mutations, and missense mutations. All patients presented within the first 2 years of life with poor vision, nystagmus, or for screening due to history of an IRD in one or more siblings (Table). Visual acuity ranged from 20/40 to light perception (LP) with several examples of marked interocular asymmetry (Table). Refractive errors were <6D spherical, <2D astigmatic as measured with retinoscopy in the youngest patients (P1 and P2) and with manifest refraction in the rest. There were no cataracts. Kinetic perimetry with a large (Goldmann size V-4e) target performed in 19 patients demonstrated oval-shaped or circular fields that extended to 5–108 of eccentricity along the meridian with the largest extent; a smaller target (I-4e) was not detectable (Table). Full-field ERGs when recorded at the initial evaluation were reported as undetectable or severely reduced in amplitude in both eyes (Table). Fundus findings confirmed previous reports. There were midperipheral pigmentary changes in all patients ranging from localized, often paravascular, depigmentation or sparing of the FIGURE 1. En face multimodal imaging in representative patients. RPE with minimal intraretinal pigment migration (Fig. 1, P9; Composite fundus photos are shown to the left; white squares outline Supplementary Fig. S1), to an overt pigmentary retinopathy the region imaged with NIR-REF and NIR-FAF shown as magnified with bone spicule pigmentation within the macula (Fig. 1, images to the right in each patient. Insets in P9 are NIR-REF and NIR- FAF images from a normal subject. point to P17). Waxy optic nerve pallor and various degrees of vascular Arrows on fundus images better preserved RPE. Arrows on NIR imaging point to peripapillary attenuation were seen in all patients. There were macular area of relative RPE preservation. Asterisks on NIR-FAF indicate the changes in all patients. In milder disease, there was obvious location of the foveal center determined with the help of SD-OCT central depigmentation that contrasted with a better appearing cross-sections. Round dark area inferior to the nerve in P17 is a large RPE in midperipheral retina (Fig. 1, P9, arrows). Peripheral RPE vitreous opacity. Arrowheads point to an example of an intraretinal changes or obvious intraretinal pigment migration are not pigmentary lesion that is hyperautofluorescent on NIR-FAF. confluent but appear to be surrounded by better appearing retina with brownish RPE (Fig. 1, P17; Supplementary Fig. S1, preservation of the NIR-FAF signal was observed in the P8). In some patients without macular atrophy the foveal peripapillary region confirming previous reports (Fig. 1; center appeared more yellowish than the coloration expected Table).8,13,28,29 There was no obvious association between for the macular pigment, and there was RPE mottling (Fig. 1, the degree of the abnormalities on ophthalmoscopy and the P9; Supplementary Fig. S1). Patients with more advanced patients’ VA, with the exception of patients with obvious macular disease showed parafoveal areas of depigmentation in foveal chorioretinal atrophy with associated poor VA (Table). a bullseye configuration or overt atrophy and pigmentation (Fig. 1A, P17). In some, there was posterior displacement of the atrophic macula in a pseudo-colobomatous configuration Severe Central Retinal Abnormalities (Table). The central retinal structure was always abnormal as exempli- NIR-FAF imaging proved to be a comfortable way of fied by representative patients in Figure 2. The youngest examining the topography of the central retinal abnormalities subject in this cohort showed severe central retinal abnormal- in this young group of patients. A NIR-FAF image in a normal ities with marked ONL thinning (Fig. 2, P1). The ONL is nearly subject shows the expected gradient of normal autofluores- undetectable within a thinned foveal center but becomes cence with greatest signal intensity near the foveal center due visible in the parafovea as a dark band riddled with punctate to the greater melanin density and taller RPE cells at the fovea, hyperreflectivities having a more normal appearance in and a decline in intensity to a homogenous grayish background peripapillary retina. The ellipsoid zone (EZ) and the interdig- with increasing eccentricity (Fig. 1, NIR-FAF, inset). NIR-FAF in itation (IZ) bands are not visible for most of the scan with the the patients showed generalized central hypoautofluores- exception of faint discontinuous signals vitread to the RPE (Fig. cence, surrounded by numerous hyperautofluorescent lesions 2, arrows) that may represent remnants of the EZ and/or IZ in that colocalized with bone spicules and with hyperautofluor- nasal pericentral and peripapillary retina. The external limiting escent lesions within that corresponded with parafoveal areas membrane (ELM) is not clearly visible. Similar patterns are seen of hyperpigmentation (Fig. 1, P17, arrowheads). In most in other patients without an obvious relationship to age. RPE patients (except P1, P2, P3, P15), an area of relative depigmentation was associated with well-defined, round

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zation of the fovea. Foveal thickness was not related to the level of visual acuity (r ¼ 0.05). Quantitation of retinal thickness parameters from horizontal cross-sections from all patients confirmed overall retinal thinning, which was most pronounced within the central 2 mm of eccentricity and less severe at greater eccentricities from the foveal center, especially in peripapillary retina (Fig. 3B, left panel). The inner retina was remarkably thick, likely a consequence of inner retinal remodeling documented in other IRD (Fig. 3B, middle panel).14,26,27,34,35 There was detectable but severely thinned outer retina in all patients (Fig. 3B, right panel). Outer retinal thinning was most severe at the most central locations, as illustrated by a greater separation of the patients’ average outer retinal thickness (Fig. 3B, right panel, dark thick trace) from the normal range within 2 mm of eccentricity than at more peripheral locations. There was no obvious relationship between the patients’ age and the extent and severity of the abnormalities (Fig. 3B). Central disease severity as judged by their VA and foveal thickness also did not relate well with the specific genotype. Patients with missense mutations (P7, P9, P10) did not differ from slightly older patients (P11, P12, P16, P19) with bi-allelic null mutations (Fig. 3; Table). Longitudinal reflectivity profiles (LRPs) generated from the transitional zone of structural change in peripapillary retina of patients with less severe disease (P14 and P9) served to identify the sequence of abnormalities in the outer retina. All outer retinal signal peaks are present in the locations near the optic nerve, most obvious in P14 (Fig. 3C, LRP ‘a’). A short distance from this location (closer to the fovea), the IZ becomes undetectable and there is only a faint EZ in both patients (Fig. 3C, LRPs ‘b’ and ‘c’). Of note, there is minor thickening of the INL as the ONL thins with increased distance from the nerve toward the fovea and no obvious change in thickness of the retinal nerve fiber layer (RNFL) in this small region. LRPs from all patients were examined to determine the location and extent of any detectable segment of the EZ band. All but the oldest patient in this series (P21, age 17) showed signals in the outer retina that were superficial to the RPE/BrM band but deeper than the ONL consistent with EZ and/or IZ, suggesting the possible FIGURE 2. Central retinal structure in RDH12-associated retinal existence of abnormal but detectable photoreceptor inner and degeneration. Nine mm-long, non-straightened, SD-OCT cross-sections possibly outer segments (POS) within the central retina of along the horizontal meridian through the fovea in representative these patients (Fig. 3D). patients. Nuclear layers are labeled. NIR-REF images are shown to the left of the SD-OCT cross sections; green lines superimposed on the images indicate the approximate location of the scans. Scans follow a Severe Central Retinal Dysfunction but Relatively temporal (T) to nasal (N) direction. Diagonal arrows point to locations Spared Extramacular Rod Function with signals superficial to the RPE that may correspond to remnants of EZ and/or IZ. Bracket in P2 delimits area of increased posterior Fixation stability was adequate in at least one eye of five backscatter due to RPE depigmentation. Asterisks positioned under- patients (P12–P14, P17, P20) to perform automated static neath the RPE mark interruptions of the RPE signal band and/or perimetry. A horizontal light-adapted sensitivity profile in P20 posterior displacement. exemplifies the results (Fig. 4A). There was detectable ONL on SD-OCT throughout the central cross-section although the hyperreflective lesions on NIR-REF and posterior backscatter- lamination of the outer retina was very abnormal. As in the ing on SD-OCT (Fig. 2, P2, bracket). Outer retinal tubulations majority of the patients, faint, interrupted linear signals above (ORTs) were observed at the peripheral border of areas of the RPE suggested the possible presence of inner and outer central RPE depigmentation, which showed interruption of the segments, although a clear outer lamination with intact ELM, RPE and posterior displacement of the ocular layers as a EZ, or IZ was not observed. Locations near the foveal center and peripapillary retina had better organization (Fig. 4A, green pseudo-coloboma in four patients (Fig. 2, asterisks; arrows). Light-adapted sensitivity to an achromatic, large Table).15,16,30–33 (Goldmann, size V) stimulus near the foveal center was Peripapillary retina with preservation of the NIR-REF signal reduced by 0.7 log units compared to the normal mean (mean showed similarly shaped areas of NIR- and SW-FAF as 6 2SD¼ 38 6 4 dB) and becomes nondetectable within 2 mm exemplified in P14 (Fig. 3A). There is an abrupt transition of the foveal center; sensitivity was again measurable near the zone (TZ) where the FAF for both excitation lights vanishes optic nerve in nasal retina coinciding with a return to a better corresponding to the location where the IZ signal on SD-OCT laminated retina (Fig. 4A, green arrow). The remaining patients becomes undetectable (Fig. 3A, yellow arrow). A small island (P12–P14, P17) had measurable sensitivities only near the of foveal autofluorescence, most noticeable on NIR-FAF, co- foveal center with maximal sensitivities ranging from 18 to 31 localizes with remnants of the EZ/IZ (Fig. 3A, white arrow) and dB (mean ¼ 24 dB). Photoreceptor mediation was assessed supports 20/40 acuity, despite the severe structural disorgani- with dark-adapted, two-color, automated perimetry, which

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FIGURE 3. Quantitative analysis of the retinal structure in RDH12-associated retinal degeneration. (A) En face retinal image in a representative patient with the mildest abnormalities in pericentral retina. Inset is a NIR-REF image showing the position of the SD-OCT scan shown in the right panel (green line). Yellow arrow points to the location that corresponds to the temporal edge of the island of peripapillary fundus autoﬂuorescence. White arrow denotes presence of disorganized but detectable outer retinal laminae, remnants of EZ/IZ, at the foveal center. (B) Total retina, inner retina and outer retinal (from OPL to RPE/BrM) thicknesses plotted as a function of eccentricity for all patients shown. Lines are color by age-group (green ¼ 2–6 years; dark red ¼ 7–11 years; red >11 years. Only one eye for each patient is shown for clarity. Thick black line is the average thickness for the parameter from all patients. Gray bands: normal limits (mean 6 2 SD) obtained from a group of young normal subjects (n ¼ 32; mean age ¼ 15 years; range, 2–24 years). (C) Magniﬁed 1 mm SD-OCT horizontal cross-section that straddles a TZ in peripapillary retina of two patients; the direction of the scans is from nasal (N) (close to the nerve) to temporal (T) retina (closer to the fovea). Overlaid traces are three LRPs positioned every ~300 lm and located on the nasal side of the TZ (‘a’), at the TZ where outer photoreceptor laminae change (‘b’), and temporal to this region (‘c’) where there is thinning and loss of outer retinal laminae. Colored segments denote the extent of the signal trough that corresponds to the ONL (blue) and the distance from the EZ or ELM signal peak to the apical RPE/BrM. (D) Lateral extent (horizontal green bars) and location of sections of the central retina with detectable albeit interrupted EZ band for each patient (vertical axis). Patients’ ID are sorted by age.

confirmed cone mediation and no detectable rod function The TPLR was used to objectively explore this finding. In a within the central retina to this stimulus (>6 log units of rod normal subject, the TPLR can be detected at very low light 2 sensitivity loss). In contrast, dark-adapted FST sensitivity levels (mean 6 2SD¼3.35 6 0.18 log scot-cd.s.m ). estimates to two color stimuli in 18/21 patients who were Increasing stimulus intensity leads to an acceleration of the old enough to perform the test (i.e., except P1 and P2), or constriction phase of the response, an increase in peak reliably complete the test (P5 was unreliable; interest response amplitude, and a retardation of the dilation phase (Fig. 4D).22 At low intensities, the pupil rapidly redilates, variability >30 dB), showed rod-mediated sensitivities that whereas at the highest intensities there is a ‘‘tonic’’ contraction were reduced on average by 2 log units (mean 6 2SD 33 ~ ¼ that lasts for a few seconds. TPLR responses elicited by a pair of 6 8 dB) compared to young normal subjects (54 6 4.6 dB, n dim, scotopically matched blue and red stimuli elicit TPLR ¼ 8, age range ¼ 11–18 years; Fig. 4B). The FST sensitivity responses that are matched in waveform (Fig. 4D, red and blue estimates were remarkably similar in both eyes (interocular traces). In a representative patient, TPLR threshold responses difference ¼ 1 6 8 dB; Fig. 4C). The findings suggest regional were first detected with a brighter stimulus near 0.5 scot- (central and midperipheral) predilection for cone and rod cd.s.m2. As in the normal subject, increasing intensity of lights disease and relative preservation of the foveal cones and leads to an increase in the amplitude of the responses and a extramacular rod function.28 similar change in morphology (Fig. 4D). TPLR responses to

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FIGURE 4. Structural–functional relationships in RDH12-associated retinal degeneration. (A) 9 mm-long, non-straightened, SD-OCT cross-sections along the horizontal meridian through the fovea in a patient. Bar above the scan show psychophysically determined cone (light-adapted, white stimulus) sensitivities. Dotted line above bar defines lower limit (mean 2 SD) of sensitivity for normal subjects. A sensitivity scale is at the top left of the panel. Cone photoreceptor mediation of the stimulus was confirmed with two color dark-adapted perimetry.25 T, temporal retina; N, nasal retina. Calibration bar to the bottom left. Horizontal green bars denote sections of the scan where a line or band can be seen above the apical RPE signal which may correspond to remnants of outer segments. Arrows point to locations where outer photoreceptor laminae (ELM, EZ, IZ) are less disorganized and are detectable as pockets that bear a resemblance to the normal lamination pattern. Asterisks denote intraretinal hyperreflectivities with posterior blocking of the SD-OCT signal consistent with central intraretinal pigment migration. (B) FST sensitivity estimates to dark-adapted spectral stimuli (blue, 467 nm; red, 637 nm) in 18/21 patients that were able to perform the test. Dashed line is the lower limit (mean2 SD) of the sensitivity to the short wavelength 467 nm stimulus in normal subjects, dotted line is the lower limit for the 637 nm stimulus. Patients’ ID are sorted by age. Only right eye shown for clarity. (C) Interocular difference of the dark-adapted FST estimates for each stimulus condition (blue, 467 nm; red,637nm).Diagonal line is the equality line; dashed lines represent the limits (6 2 SD) of the interocular differences. (D) Families of dark-adapted TPLR waveforms elicited with brief (100 ms), blue (467 nm) stimuli delivered over the intensity range of 3.75 to þ4.25 log scot-cd.s.m2 (0.5 log unit steps) on the same stimulator unit used for FST measurements. Traces plot the horizontal pupil diameter as a function of time in a normal subject compared to a representative patient. Overlapping thick red traces near TPLR threshold are responses elicited by red (637 nm) stimuli scotopically matched to the blue stimuli (blue traces). Stimulus monitor shown as a vertical bar at bottom left.(E) FST sensitivity loss plotted against TPLR sensitivity loss for each eye of patient who were able to complete both tests (n ¼ 18). Symbols with black outline ¼ right eye; gray ¼ left eye. Diagonal line is the equality line.

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scotopically matched blue and red stimuli were grossly ity estimates in the patients were similar to reports in older matchedinwaveform,suggestingrodmediationofthe subjects and not significantly different to estimates from a small pupillary light reflex as in the normal subjects (Fig. 4D). The group of normal children from this study.21,23 It remains waveform shape at threshold is similar to that of the normal possible that young subjects may respond conservatively, albeit subject at threshold, suggesting an insensitive, rod-driven TPLR reproducibly, to the FST task and overestimate the sensitivity with normal kinetics.22 TPLR thresholds in the patients were loss, a topic that deserves further study. The TPLR as measured elevated by an average 2 log units (1.1 6 0.5 log scot- in this study was initially described in cohorts of patients and cd.s.m2) compared to normal subjects (3.4 6 0.2 log scot- normal subjects that included children,22 but there is still a cd.s.m2).The difference in TPLR thresholds between these need to further define the reliability of the methodology in two spectral stimuli in the patients (0.2 6 0.7 log scot- pediatric populations, a factor that may help explain the minor cd.s.m2) fell well within the range expected for rod discrepancies in the sensitivity estimates between the two photoreceptor mediated responses in normal subjects (0.5 methodologies. 6 0.4 log scot-cd.s.m2). Sensitivity losses estimated with FST does not provide information about the topography of TPLR related well with losses determined with FST (r ¼ 0.83; the visual field, which we could document only in a small Fig. 4E). group of patients with static perimetry. There were sectors in the periphery of some of our patients that showed a better appearance on en-face imaging. It remains to be determined DISCUSSION where in the extramacular retina the residual rod function originates and whether these better-appearing regions mediate Homozygous mutations in RDH12 have been associated with the residual rod function as has been done in other EORD.53 LCA, EORD, or autosomal recessive retinitis pigmentosa, and 3,5,8,13,30,32,36–44 There is also the possibility of rod-mediation within the central rarely with cone rod dystrophy (CRD). There retina in regions not explored by the horizontal sensitivity are examples of segregation of RHD12 mutations in an profile used in this work. While the overall topography of the autosomal dominant fashion and several heterozygous muta- 6,8,32,36,37,39,42 retinal dysfunction awaits confirmation in larger groups of tions have been reported in patients with RP. patients with RDH12-IRDs, the pattern observed in this work, Instead of the phenotypic diversity implied by the different taken together with those of previous reports, strongly diagnoses used to describe the autosomal recessive disease, suggests that autosomal recessive mutations in RDH12 cause review of the literature reveals a consistent phenotype a predominantly severe central retinal degeneration that is characterized by a midperipheral pigmentary retinopathy of 32,35,54–57 3,8,13,28,36,37,45 5,8,13,28,30,33,38–41 consistent with an early CRD phenotype. Reports of early onset and a maculopathy CRD phenotypes in milder forms of -IRD supports a 15,16,30–33 RDH12 that includes macular pseudo-colobomas. Whereas common disease expression with a wide spectrum of severity there is a wealth of information on the retinal structure of the in RDH12-IRD.32,44,55 retinal degeneration associated with RDH12 mutations, details The disease mechanisms in RDH12-IRD are not fully of the associated retinal dysfunction are comparatively understood, but the severity and early presentation of the 13,14,30,31,36,44 scarce. Previous studies that have used electro- phenotype that results suggest a critical role in human retinal retinography have reported severely abnormal or undetectable physiology. RDH12 localizes to the rod and cone photorecep- responses and functional data other than visual acuity is tor inner segment and is able to reduce all-trans- and all-cis 14,30–32 limited. In this study, we used multimodal imaging, retinoids.11,58,59 Murine Rdh12 knockouts, however, do not visual psychophysics, and dark-adapted chromatic pupillom- show an overt retinal degeneration or major slowing of the etry to describe the retinal structure and function of pediatric visual cycle.58,60,61 Redundancy of retinal dehydrogenases, patients with homozygous or compound heterozygous specifically RDH8, has been offered as an explanation for this mutations in RDH12. We found severe and early macular discrepancy or a different role of RDH12 in humans compared disease in all our patients, confirming previous observa- to the murine model.60 An alternative role in clearing all-trans- 5,8,32,36–40,42,43 tions. The changes were obvious by SD-OCT in retinal from rod and cone inner segments, in the movement of our youngest patient (P1, age 2) who had an otherwise retinoids, or in the detoxification of lipid peroxidation benign fundus appearance and was asymptomatic. products, has been suggested. Removal of all-trans retinal We confirmed severe central cone and rod dysfunction would prevent this photosensitizer from damaging photore- accompanying the severe central structural abnormalities in ceptors.62 The pattern of disease expression documented in association with comparatively milder, presumably extramac- this work suggests vulnerability of both rods and cones within ular rod dysfunction as assessed with FST and two-color static the central retina and relative peripheral rod preservation.28 perimetry, and confirmed by dark-adapted chromatic TPLR.22 Similar patterns have been reported in at least two other forms The TPLR responses to relatively dim and brief (100 ms) stimuli of LCA/EORD, AIPL1-LCA, and CRB1-LCA.53,63–65 Coinciden- near threshold in the patients are inconsistent with nonclas- tally, in these three molecular subtypes, a role of the encoded sical photoreception mediation of the pupillary light reflex, proteins in photoreceptor maintenance has been suggested known to co-exist or replace classical photoreception in cases with light exposure being a possible common pathogenic of EORD.46 We could not confirm, however, if the level of factor.53,63,66 It has been suggested that the preservation of peripheral function was proportionally reduced for cone and peripheral rod function in in LCA-CRB1, for example, may be rod function as the FST sensitivities for both short- and long- related to the survival of peripheral, rod-dominated retina, in wavelength stimuli were mediated by rods in all patients.21 The the shadow of the iris and ciliary body in this and other light- use of alternative methods of cone isolation will help answer sensitive retinopathies.53 this important question.47–49 The reliability of the sensitivity The peculiar preservation of the peripapillary retina in estimates in this group of young patients was also considered. RDH12-LCA, PRPH2-IRD, and ABCA4-IRD has been suggested Normal age-related changes in rod sensitivity may not be to result from light protection conferred by the ‘‘shadow’’ of an expected to occur in children within the age range represented overlying thick retina.13,67–69 However, in this study, we did in this study.50–52 FST and TPLR thresholds related well, but the not find a relationship between the thickness of the overlaying relationship was not perfect with examples of TPLR showing RNFL or inner retina and the preservation of the outer retina in less sensitivity losses compared with FST measures. FST peripapillary retina, arguing against this mechanism. The area thresholds were considered reproducible. Within-visit variabil- of peripapillary preservation tended to extend just outside of

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the vascular arcades and did not match exactly the expected dysfunction to be expressed clinically in a uniformly severe direction of the RNFL bundles, which correspond to the phenotype, as characterized in this work. thickest inner retina, also standing against this hypothesis.34,70 Limited longitudinal studies and multiple cross-sectional Mapping the topography of each of the individual retinal layers observations in patients with RHD12-associated IRD indicate should help answer this question, a task that may be visual acuity may be relatively preserved early in the course of complicated by unstable fixation in severe RHD12-IRD. the disease, while a somewhat steep decline occurs in the Intraretinal differences in expression of protective factors, second decade of life, reaching count finger or LP level of photoreceptor densities, photoreceptor/RPE ratios, or in the vision by age 20.3,8,14,28–32,36,41,78 Relatively preserved visual movements of retinoids between glia and/or the vascular acuity and even color vision, may be documented in RDH12- compartment and the retina may also be at play.71–73 RPE cells IRD despite severe central retinal disease and is still in keeping deliver retinosomes, retinyl esters-containing lipid droplets with a diagnosis of CRD.32,37 Such pattern of early relative thought to be involved in the storage and/or trafficking of preservation of visual acuity and then rapid loss is reminiscent retinoids between the RPE and photoreceptors. These of numerous maculopathies that show preservation of visual organelles may ensure a steady supply of 11-cis retinal to acuity despite severe central retinal disease.79–84 Thus, visual photoreceptors despite fluctuations in dietary vitamin A.74 In acuity in this condition, as in most IRDs, is not a reliable vitro site-directed mutagenesis that abolishes the oxidoreduc- indicator of the severity of the central retinal phenotype, tase activity of ENV9, an ortholog of human RDH12, suggests a although it is critical to the patients’ quality of life.27,78 Once role for the enzyme in the formation of lipid droplets.75 It is the central island is compromised a rapid decline of the possible that the central retina may be particularly susceptible patients’ VA may be expected.31 In RDH12-IRD the surround- to degeneration due to higher demands for retinoids, ing pericentral retina, however, may not be able to sustain particularly by cone photoreceptors.16,76 usable or rehabilitable central vision as is the case in other Most variants reported in the current work are predicted to macular diseases, such as Stargardt’s disease. The hope is that cause lack-of-function proteins. The most common mutation residual peripheral rod vision at that stage may be enough for was Ala269del (>60%; 13/21 of our patients), which is a ambulation, a pattern observed in patients with comparatively frameshift mutation leads to a premature stop codon in exon 6 less severe CRDs and consistent with residual peripheral and a truncated, nonfunctional protein.3,8,28,30,63–65 In vitro temporal islands reported in young patients with RDH12- studies have shown that this mutation results in defective IRD.30–32,35 The concern is that severe peripheral rod disease enzymatic activity, impairing the conversion of all-trans retinol at that stage in RDH12-IRD may not serve patients well due to from all-trans retinal. The second most common mutation was the depth of the peripheral dysfunction and that total Arg295Stop (33%; 7/21 patients), a null mutation in exon 7 at blindness will result. the tail end (last 21 amino acids) of the protein.13,14,37,77 The EORDs remain excellent platforms where the risk/benefit mutation likely leads to nonsense-mediated RNA decay and ratio for vision change after gene therapy may be stacked in either a nonfunctional or absent protein. The third most favor of visual gain. Today, targets of treatments in the EORD common variant was Val233Leu (24%; 5/21 patients), a group are somewhat biased toward genetic subtypes that missense change in exon 6 predicted to be ‘‘neutral.’’ A show structural–functional dissociations (for example previous study, however, deemed this pathologic based on RPE65, CEP290, GUCY2D).85–90 These conditions may be predictions of the effects of coding nonsynonymous variants simpler to treat as the expectation is that treatments of on protein function using the ‘‘Sorting Tolerant From relatively preserved retina with a disproportional dysfunction Intolerant’’ (SIFT) algorithm.28 Four out of the five patients should theoretically lead to measurable gains in vision in the with this variant had the stop codon mutation Ala269del in the short-term supporting efficacy, as it did in RPE65-LCA trials. other allele; the remaining patient (P9) had a Ser134Pro, which However, such structural–functional dissociation phenotype has not been reported. This variant is predicted to be damaging constitutes a small fraction if the entire field of inherited by RESCUE-ESE (http://genes.mit.edu/burgelab/rescue-ese/, retinal degenerations is viewed as a whole. There is a need available in the public domain). The fact that other codon for a different approach to the alternative scenario where the 233 missense variants (Val233Asp) have been reported and retinal degeneration does not follow clear functional– that the Val233Leu variant reported in this work segregate with structural dissociations or where the disease may be locally the disease in four affected individuals from a large family (13 severe as it occurs in RDH12-IRD. Treatments of forms of siblings), implicate this specific variant as disease causing.13,28 LCA/EORD that are caused by defects in photoreceptor Studies are in progress in our laboratory aimed to determine proteins such as RDH12-LCA also promise to move the target whether the Val233Leu or Ser134Pro variants affect protein of interventions from the retinal pigment epithelium (RPE)— folding or function. the primary site of diseases such as RPE65-LCA—into the RDH12-IRD patients included in this study showed early photoreceptor but, more importantly, from simpler mecha- and severe phenotype without an obvious relationship with nisms of disease to hopefully offer therapeutic solutions of the patients’ age. Comparisons between patients with mis- complex causes of vision loss where epigenetic modifiers of sense vs. null mutations revealed no major differences in the disease expression, such as light exposure, may play a severity of the phenotype as judged by age at presentation and role.10,91–94 In this work, we present evidence of retained severity of the structural or functional changes (Fig. 4; Table). photoreceptors within the central retina in all patients Convergence into a homogeneously severe phenotype at an examined, which raises hope that correction of the genetic early age may blur possible genotype–phenotype differences in defect may lead to arrest of the degeneration and hopefully to severity that may have existed very early in life and may also improvements in vision. The absence of clear SD-OCT signals explain the apparent lack of relationship between age and the that correspond to preserved POS within the central retina of severity of the phenotype in this young group of patients. even our youngest patient is concerning, and gene augmen- Studies in larger groups of patients representing a broader age tation trials should consider and discuss with patients and range are required to better determine the relationships families the fact that the outer segment may be structurally between the phenotype and genotype and between disease impaired beyond repair. The hope is in the alternative severity and age. Alternatively, RDH12 mutations—even possibility, that gene augmentation may lead to restoration of potentially milder missense mutations—may cause sufficient the structure of the POS and vision as is known to occur

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following correction of various insults to this important 10. Chen C, Thompson DA, Koutalos Y. Reduction of all-trans- structure.95–99 retinal in vertebrate rod photoreceptors requires the com- Although there may be examples of milder phenotypes, the bined action of RDH8 and RDH12. J Biol Chem. 2012;287: majority of patients with RDH12-IRD in this and previous 24662–24670. reports share a severe, early disease, which suggests we may 11. Haeseleer F, Jang G-F, Imanishi Y, et al. Dual-substrate have no choice but to move the window for potential specificity short chain retinol dehydrogenases from the treatments to younger ages when there is still treatable retina, vertebrate retina. J Biol Chem. 2002;277:45537–45546. raising new risk/benefit concerns. Early clinical trials that focus 12. Hofmann L, Tsybovsky Y, Alexander NS, et al. Structural on safety are expected to include patients with the most severe insights into the Drosophila melanogaster retinol dehydro- abnormalities, and it can be anticipated that results in terms of genase, a member of the short-chain dehydrogenase/reduc- efficacy may be modest. Careful consideration of the outcomes tase family. Biochemistry (Mosc). 2016;55:6545–6557. chosen for future clinical trials and careful interpretation of 13. Garg A, Lee W, Sengillo JD, Allikmets R, Garg K, Tsang SH. early results of those interventions will be needed to prevent Peripapillary sparing in RDH12-associated Leber congenital that a treatment, such as gene augmentation, is prematurely amaurosis. Ophthalmic Genet. 2017:38:575–579. deemed ineffective depriving less severely affected patients 14. Jacobson SG, Cideciyan AV, Aleman TS, et al. 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