Disease Boundaries in the of Patients with Usher Syndrome Caused by MYO7A Mutations

Samuel G. Jacobson,1 Tomas S. Aleman,1 Alexander Sumaroka,1 Artur V. Cideciyan,1 Alejandro J. Roman,1 Elizabeth A. M. Windsor,1 Sharon B. Schwartz,1 Heidi L. Rehm,2 and William J. Kimberling3

PURPOSE. To study retinal microstructure in Usher Syndrome he Usher syndromes (USH) are a molecularly heteroge- type 1B (USH1B) caused by MYO7A mutations as a prelude to Tneous group of autosomal recessive diseases with dual treatment initiatives. sensory deficits of impairment and retinal degenera- tion.1 The USH encode of different classes. A METHODS. Patients with MYO7A-USH1B (n ϭ 17; ages 5–61) unifying hypothesis is that USH gene products are part of a were studied with optical coherence tomography. Retinal lam- network in the region of the cilium of the photorecep- inae across horizontal and vertical meridians were measured. tor. Dysfunction of this network is thought to lead to the Colocalized visual sensitivity was measured with automated retinal degeneration.2,3 perimetry to enable comparisons of function and structure in USH1B is the most common form of USH1 and is caused by the transition zones. mutations in MYO7A, a gene encoding an unconventional 4–6 RESULTS. Laminar architecture of the central retina in MYO7A- . Recent promise of somatic retinal in USH1B ranged from normal to severely abnormal. Within the MYO7A-USH1B7 prompts questions about the feasibility of transition zone between normal and abnormal retina, the first treatment in humans. Translation to clinical trials is compli- detectable abnormality was an increase in prominence of the cated by the fact that there is currently no Myo7a-deficient OLM (outer limiting membrane). Declining ONL thickness was animal model with retinal degeneration approximating the 1 accompanied by increased thickness of the OPL and normal or human condition. Dual localization of the gene product in retinal pigment epithelium (RPE) and photoreceptors adds increased INL. Undetectable ONL and OPL and hyperthick INL 1,4,8 were features of severe at further eccentricities further complexity to any strategy for gene transfer. The into the transition zone. Visual sensitivity in the transition zone onus falls on studies of molecularly defined patients to clarify declined with the decrease in ONL thickness. the detailed retinal of MYO7A-USH1B and seek clues about disease mechanism that will enable translation. CONCLUSIONS. Patients with MYO7A-USH1B can have regions of Recently, we embarked on studies of human MYO7A- structurally and functionally normal retina with definable tran- USH1B to quantify abnormalities in the sitions to severe laminopathy and visual loss. The earliest de- layer and RPE. The pattern of results in MYO7A-USH1B was tectable structural markers of disease may represent Mu¨ller compared with those in other USH genotypes, for which there glial cell response to photoreceptor stress and apoptosis. Vi- was experimental evidence of localization of gene products sual losses were predictably related to a decline in ONL thick- only to photoreceptors. We concluded that MYO7A-USH1B ness. The prospect of focal treatment of MYO7A-USH1B, such was behaving phenotypically like these other photoreceptor as subretinal gene therapy, prompts the need to identify retinal diseases.9 The present study seeks further details about the locations that warrant consideration for treatment in early retinopathy resulting from human MYO7A mutations. Topo- phase trials. The transition zones are candidate sites for treat- graphical maps of photoreceptor and inner retinal laminae ment, and laminar architecture and visual sensitivity are possi- indicate that there are not only photoreceptor layer losses but ble outcomes to assess safety and efficacy. (Invest Ophthalmol also inner retinal abnormalities. The transition zone between Vis Sci. 2009;50:1886–1894) DOI:10.1167/iovs.08-3122 abnormal and normal-appearing retina, an area worth consid- ering for focal treatment in a phase I trial, was investigated with high-resolution, cross-sectional imaging and visual thresh- olds. From the 1Department of , Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania; the 2Depart- ment of , Harvard Medical School, Boston, Massachusetts; METHODS and the 3Usher Syndrome Center, Boys Town National Research Hos- pital, Omaha, Nebraska. Human Subjects Supported by grants from the National Neuroscience Research There were 17 patients with USH1B caused by MYO7A mutations Institute, The Foundation Fighting Blindness, the Macula Vision Re- (Table 1). Patients underwent a complete eye examination. Normal search Foundation, Hope for Vision, the Chatlos Foundation, Research subjects for optical coherence tomography (OCT; n ϭ 33; ages 5–58 to Prevent Blindness, and the Ruth and Milton Steinbach Fund. years) and for psychophysical testing (n ϭ 9; ages 19–48 years) were Submitted for publication November 6, 2008; accepted February 24, 2009. also included. Informed consent was obtained for all subjects; proce- Disclosure: S.G. Jacobson, None; T.S. Aleman, None; A. dures adhered to the Declaration of Helsinki and were approved by the Sumaroka, None; A.V. Cideciyan, None; A.J. Roman, None; E.A.M. institutional review board. Windsor, None; S.B. Schwartz, None; H.L. Rehm, None; W.J. Kim- berling, None Optical Coherence Tomography The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- Retinal cross-sections were obtained with OCT. Data were acquired in ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. 15 of the patients with Fourier-domain (FD) OCT imaging (RTVue-100; Corresponding author: Samuel G. Jacobson, Scheie Eye Institute, Optovue Inc., Fremont, CA). Two patients had imaging with either University of Pennsylvania, 51 N. 39th Street, Philadelphia, PA 19104; OCT3 (F7, P1) or OCT1 (F9, P1) (Carl Zeiss Meditec, Inc., Dublin, CA). [email protected]. The principles of the method and our recording and analysis tech-

Investigative Ophthalmology & Visual Science, April 2009, Vol. 50, No. 4 1886 Copyright © Association for Research in Vision and Ophthalmology

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10,12,13,16 TABLE 1. MYO7A Mutations in Patients with USH1B flective layer continuous with the INL. At the boundary be- tween the ONL and the INL, there is a hyperreflective signal that corresponds to the outer plexiform layer (OPL).10 OPL thickness was Family Patient Sex/Age (y) Change(s) defined as the distance between the minima and maxima of the signal slopes on either side of the local signal maxima at the boundary 1 1 F/4 His1355fs between the ONL and the INL. In normal subjects, the signal corre- Arg2024X 2* 1 M/6 Tyr333X sponding to the retinal pigment epithelium (RPE) was assumed to be Tyr333X the most sclerad peak within the multipeaked scattering signal com- 2 M/8 Tyr333X plex,10 deep in the retina. In abnormal , the presumed RPE peak Tyr333X was sometimes the only signal peak deep in the retina; in other cases, 3 1 F/11 Ala1770Asp it was apposed by other major peaks. In the latter case, the RPE peak 19-2AϾG was specified manually by considering the properties of the backscat- 4* 1 M/17 Glu495X tering signal originating from layers vitread and sclerad to it.9 The Cys31X presence of photoreceptor inner/outer segment signal and outer lim- 5 1 F/19 Arg634X iting membrane signals vitread to the RPE peak were also assessed.9 Gly1982Glu 2 F/21 Arg634X For topographic analysis, the precise location and orientation of Gly1982Glu each scan relative to retinal features (blood vessels, intraretinal pig- 6 1 M/21 Gly955Ser ment, and optic nerve head) were determined on the video images of 2187ϩ1GϾA the fundus. LRPs were allotted to regularly spaced bins in a rectangular 7 1 M/24 Glu968Asp coordinate system centered at the fovea; the waveforms in each bin Lys164Asn were aligned and averaged. For two-dimensional maps, 0.3-mm2 bins 8 1 M/30 Tyr2015His were used for sampling, whereas 0.15-mm2 bins were used for analysis Asp2010Asn along the horizontal and vertical meridians. Overall retinal, ONL, and 9* 1 F/34 Arg1240Gln inner retinal thicknesses were measured. Missing data were interpo- Arg1240Gln 10* 1 M/35 Arg669X lated bilinearly, thicknesses were mapped to a pseudocolor scale, and Arg1591fs the locations of blood vessels and optic nerve head were overlaid for 11* 1 F/39 Leu1858Pro reference.12,14,16 Glu1716X 12* 1 F/41 Ala1288Pro Visual Psychophysics Arg1743Trp 17 2 F/46 Ala1288Pro Static threshold perimetry was performed and analyzed as published. Arg1743Trp Static thresholds were determined with 1.7°-diameter, 200-ms duration 13* 1 M/44 Gly163Arg white stimuli under light-adapted conditions. Thresholds were mea- 5921GϾT sured along the horizontal and vertical meridians (2° intervals) crossing 14* 1 F/61 Gln1798X fixation and corresponding to the same retinal regions examined with Glu1917X OCT. Sensitivity (1/threshold) for each location was compared with the mean normal data to determine -specific sensitivity loss. * Families in which some genotype and/or phenotype information has been reported in Reference 9. RESULTS 10–14 niques have been published. Briefly, overlapping OCT scans that Topographical Maps of Retinal Thickness in were 4.5 mm in length were used to cover the horizontal and vertical MYO7A- meridians up to 9 mm eccentricity from the fovea. At least three OCT Mutant Retina scans were obtained at each retinal location. Dense overlapping FD- Maps of thickness topography across a wide expanse of central OCT raster scans (101 raster lines of 512 A-scans each covering 6 ϫ 6 retina are illustrated (Fig. 1) for the full cross-section of retina mm) were performed to sample a 18 ϫ 12 mm region of the retina (left column), the ONL (middle column) and the inner retina centered on the fovea.10–14,15 A video fundus image was acquired by (right column) in a normal subject (Fig. 1A) and three patients the commercial software and saved with each OCT scan. with MYO7A-USH1B of different ages and disease stages (Figs. Post-acquisition processing of OCT data was performed with cus- 1B–1D). Normal retina has a foveal depression surrounded by tom programs (MatLab 6.5; The MathWorks, Natick, MA). Longitudinal parafoveal thickening and then a decline in thickness with reflectivity profiles (LRPs) making up the OCT scans were aligned by increasing eccentricity. Thickening at superior and inferior using a dynamic cross-correlation algorithm with a manual override poles of the optic nerve represent converging axons (Fig. 1A, when crossing structures (for example intraretinal pigment) inter- left). F5,P2, a 21-year-old patient with MYO7A-USH1B, showed rupted local lateral isotropy of signals. Repeated scans were laterally a normal pattern of retinal thickness in a large region of central aligned and averaged to increase the signal-to-noise ratio and allow retina but thickness was reduced at greater eccentricities (Fig. better definition of retinal laminae.10,14 Overall retinal thickness was 1B). F2,P1, a 6-year-old patient with MYO7A-USH1B, showed a defined as the distance between the signal transition at the vitreoreti- pattern of retinal thickness that was also similar to normal in nal interface (labeled T1 in Ref. 10) and the major signal peak corre- the more central retina, but there was loss of thickness inside sponding to the RPE.16 Two nuclear layers, the outer photoreceptor and beyond the vascular arcades (Fig. 1C). F10,P1, age 35, has nuclear layer (ONL) and the inner nuclear layer (INL), were defined in apparently increased or normal retinal thickness within the regions of scans showing two parallel stereotypical hyporeflective vascular arcades and decreased thickness at greater eccentric- layers sandwiched between the RPE and vitreoretinal interface.9,12–14 ities (Fig. 1D). The boundaries of these two hyporeflective layers were defined by the Mapping ONL and inner retinal thickness across this region minima and maxima of the signal slopes. Transition regions in which dissected the morphologic disease effects on photoreceptors there was change from two to one hyporeflective layer were present in from the effects on the postreceptoral retina. ONL topography patient scans. In all cases the single hyporeflective layer was laterally of the normal retina peaked centrally and declined with dis- continuous with the INL. An inner retinal thickness parameter was tance from the fovea; parafoveal thinning occurred more grad- defined as the distance between the signal transition at the vitreoreti- ually in the superior retina (Fig. 1A, middle). F5,P2 retained a nal interface and the sclerad boundary of the INL or the single hypore- large central island of ONL that extended more superiorly than

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FIGURE 1. Retinal thickness topography of MYO7A-USH1B. Topographical maps of total retinal (left), ONL (middle), and inner retinal (right) thicknesses in a normal 24-year-old subject (A) and three patients with MYO7A-USH1B of different ages and disease stages (B–D). Traces of major blood vessels and location of optic nerve head are overlaid on each map (depicted as right eyes). Pseudocolor scales are shown beneath the normal maps. Insets: thickness difference maps showing regions that were abnormally thin (blue), within normal limits (white, defined as mean Ϯ 2 SD), or thick (pink), compared with normal. Optic nerve outlines were schematized in both the topographic and thickness difference maps. T, temporal; N, nasal; S, superior; I, inferior, F, fovea.

inferiorly (Fig. 1B). The inset comparing the map of F5,P2 to 1D, inset). These observations led to a locus-by-locus quantita- the lower limit of normal (mean Ϫ 2 SD) shows that most of tion of the thickness parameters along horizontal and vertical this central island had normal ONL thickness (Fig. 1B, inset). meridians in our entire cohort of patients with MYO7A-USH1B There was a decrement of ONL with eccentricity and it became (Fig. 2). undetectable at greater eccentricities. F2,P1 had a relatively smaller island of preserved ONL compared with that of F5,P2. Again, there was greater superior than inferior extension (Fig. Inner and Outer Retinal Architecture in 1C). Most of the remaining ONL was within normal limits (Fig. MYO7A-Mutant Retinas 1C, inset). F10,P1 showed a smaller central ONL island and only the very center of this island retained normal thickness Cross-sectional images along the horizontal and vertical merid- (Fig. 1D, inset). ians in a normal subject and in F3,P1, an 11-year-old patient Normal inner retinal thickness topography had a foveal with MYO7A-USH1B, are shown (Fig. 2A). In the normal retinal depression surrounded by an annulus of increased thickness cross section, there was a foveal depression and the surround- and a crescent-shaped thickening extending toward the optic ing retina was laminated with prominent low-reflectivity cellu- nerve from superior and inferior retina (Fig. 1A, right). Inner lar layers (ONL and INL) and intervening hyperreflective syn- retinal topography in two patients with MYO7A-USH1B, F5,P2 aptic laminae. F3,P1 also showed a foveal depression with and F2,P1, was normal in the central retina but there was normal-appearing foveal ONL thickness. The ONL, however, increased thickness at greater eccentricities (Figs. 1B, 1C, in- diminished in thickness with eccentricity from the fovea and sets). F10,P1, who had far more advanced disease, showed became abnormally thinned or undetectable at greater eccen- abnormally increased thickness of the inner retina across the tricities. The inner retina appeared thickened at the eccentric- entire region sampled except for a small central region (Fig. ities with reduced ONL.

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FIGURE 2. Retinal laminar architecture in MYO7A-USH1B. (A) Cross-sectional scans along the horizontal (left) and vertical (right) meridians in a normal subject (top) and an 11-year-old patient with MYO7A- USH1B (bottom). OCT scans are shown in grayscale with the lowest reflectivity in black and the highest in white. Insets: schematic location of the scans. Brackets defining ONL and inner retina are labeled (left edge of the horizontal scans) and a bracket showing overall retinal thickness is at the right edge of the vertical scans. (B–D) Thickness of the retina (B), ONL (C), and inner retina (D) along the horizontal and vertical meridians in all 17 patients, identified by symbols and grouped by age (yellow, 4–30 years; red, 34–61 years). Shaded areas: normal limits; mean Ϯ 2 SD: (B) n ϭ 25; (C) n ϭ 25; (D) n ϭ 14.

Overall retinal, ONL, and inner retinal thicknesses across patients had abnormally reduced foveal ONL (Fig. 2C). A mi- the horizontal and vertical meridians were quantified for the 17 nority of the younger patients had normal ONL extending to patients with MYO7A-USH1B (Figs. 2B–2D). Patients are iden- Ͼ6 mm from the fovea. Most patients with MYO7A-USH1B, tified by individual symbols; colors distinguish younger (yel- however, had abnormally thinned ONL by 2 to 4 mm eccentric low, 4–30 years) from older (red, 34–61 years) ages. Also to the fovea, and ONL was not detectable at greater eccentric- plotted are normal limits (gray, Ϯ2 SD from mean). Retinal ities. Inner retinal thickness is normally at its minimum in the thickness shows a wide spectrum of results from normal to fovea. Parafoveal thickening, a feature of the normal retina, is abnormally reduced (Fig. 2B). ONL thickness in all the younger also present in MYO7A-USH1B. At eccentricities beyond ap- patients was normal at the fovea while many of the older proximately 3 mm eccentric to the fovea, some younger pa-

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tients show slightly increased inner retinal thickness. Nearly all imum dark-adapted white flash ranged from 10 to 73 ␮V older patients have hyperthick inner retina from paracentral to (mean, 30 ␮V; SD, 20 ␮V; normal mean, 497 ␮V; SD, 111 more peripheral regions of measurement (Fig. 2D). ␮V20). Localized visual function, specifically in the transition zone, was measured with light-adapted static perimetric pro- Details of Laminar Architecture in the Transition files, centered at the fovea and along the horizontal (Fig. 5A) Zone from Normal to Abnormal Retina and vertical (Fig. 5B) meridians. The results, shown for seven representative patients with MYO7A-USH1B, indicate that sen- Cross-sectional images from the fovea along the temporal ret- sitivity could be normal in a wide extent of central field, as in inal meridian in a normal subject and a patient with MYO7A- F5,P1 and F8,P1. A decline of visual sensitivity was noted at 6 USH1B are shown (Fig. 3A). There was an obvious transition to 7 mm eccentricity in these two patients along the temporal zone in F5,P2 from more normal-appearing retina to markedly and superior retina; inferior retinal abnormalities begin closer abnormal retina. What were some of the features of the tran- to fixation (Figs. 5A, 5B). Vertically asymmetric sensitivity pro- sition zone? There appeared to be greater prominence of the files were also present in two other patients (F4,P1 and F9,P1), hyperreflective layer considered to be the outer limiting mem- with sensitivity extending more into superior than into inferior brane (OLM) beginning at approximately 3 mm temporal from retina. There were also patients (F11,P1; F12,P2; and F12,P1) the fovea and continuing until this layer was no longer detect- with reduced sensitivity throughout the profiles, but more able at approximately 7 mm eccentricity. The ONL thinned function centrally than at greater eccentricities. with eccentricity gradually from ϳ5 to 6 mm eccentric to the Do the transitions from normal to abnormal function relate fovea until it was not discernible at distances of Ͼ8 mm. The to transitions from normal to abnormal retinal structure noted OPL, the hyperreflectivity immediately vitread to the ONL, in the OCT data analyses? Cross-sectional OCT images through appeared to increase in thickness beginning at ϳ6 mm eccen- the horizontal meridian from the fovea into temporal retina in tricity; after Ͼ7.5 mm it thinned again and became indistinct. a patient with MYO7A-USH1B (F4,P1) illustrate the structural The INL appeared nearly normal from the fovea through most changes within the transition zone and the accompanying of the transition zone but after ϳ8 mm, lamination became changes in visual sensitivity (Fig. 5C). There is normal lamina- indistinct; there may be some thickening at this eccentricity. tion, normal ONL thickness, and normal sensitivity that extend Quantitation of thickness of the ONL, OPL, and INL in F5,P2 from the fovea to ϳ2 mm into temporal retina. From 2 to 4 mm compared with normal subjects over this temporal retinal re- the retina retained lamination, but there was gradual ONL gion is shown (Fig. 3B). OLM prominence (not quantified) is thinning and loss of IS/OS and OLM signals. OPL and INL also indicated. The ONL thickness was within normal limits thickened and sensitivity declined below normal limits (dashed until approximately 6 mm eccentricity, but OLM prominence line, Fig. 5C). Between 4 and 5 mm temporally, the ONL is was evident at lesser eccentricities. As the ONL thinned to barely discernible and there is further loss (Ͼ2 log units) of below the normal limits, the OPL thickened; with increased visual sensitivity. Beyond 5 mm, there is a change in retinal eccentricity, OPL thickness returned to normal and then was structure with loss of measurable ONL and of normal lamina- not discernible. In the region of decreasing ONL and increasing tion. This structural change was accompanied by Ͼ3 log units OPL, the INL was slightly thicker than normal. of visual sensitivity loss. Subdividing the transition zone allowed us to ask if the A relationship between ONL thickness and visual function results in F5,P2 were generalizable to other patients with has been established in normal human retinas and in retinal MYO7A-USH1B. The transition zone was divided into four degenerations.9,12,14,21 We used this general function–struc- contiguous subzones. Subzones a to d are marked on the scans ture relationship to study colocalized visual sensitivity and ONL of F5,P2 and the quantitative analyses of thickness (Figs. 3A, thickness along the horizontal and vertical meridians of pa- 3B). Subzone a represented retina with abnormal prominence tients with MYO7A-USH1B (Fig. 5D). ONL thickness, expressed of the OLM and normal or nearly normal thickness of the ONL, as a fraction of mean normal thickness (in log units) for any OPL and INL. In subzone b, the ONL decreased in thickness to given location, was related to the colocalized loss of visual below the normal limits; the OPL in this region increased in sensitivity (slope, 0.3; correlation, r ϭ 0.76). Thus, in MYO7A- thickness; and the INL was slightly increased or within normal USH1B, transition zone visual sensitivity behaved as if photo- limits. The OLM varied from visible to not detectable. Subzone receptor loss is the dominant contributor to the visual dysfunc- c still showed definable retinal lamination but the ONL was tion.9 reduced to barely detectable; the OPL decreased and the INL increased in thickness. There was no visible OLM. Subzone d is 11,15,18 what we previously termed “bilaminar” retina —no mea- DISCUSSION surable ONL or OPL and a thickened hyporeflective layer that may well represent intermingled INL and residual ONL nu- USH1 has been considered to be the most severe form, based clei.13,19 mainly on the profound and vestibular involve- Representative scans with demarcation of the subzones are ment. The associated retinal disease is often described as a shown in another region of F5,P2 and in three other patients “prepubertal onset of .”6 Molecular discov- (Fig. 4A). Quantitation of ONL, OPL, and INL thickness in ery has identified many different causative genes within the examples of each of the subzones are also shown (Fig. 4B). Not clinical category of USH1, but detailed information on pheno- all subzones were detectable in each patient, but the order of type has not usually accompanied the reports of new geno- laminar architectural changes from most normal (a) to most types. Proof of concept that subretinal gene delivery may be abnormal (d) was never contravened. useful to treat USH1B caused by MYO7A mutations7 is a key reason to refine our understanding of this retinal phenotype. Visual Function in the Transition Zone The lack of a retinal degeneration component in the - of MYO7A-USH1B mutant mouse and no available human postmortem donor retinas from patients with MYO7A-USH1B makes noninvasive Full-field (ERG), the traditional measure of study of the human retinal disease essential for developing a retina-wide function, was performed (or results were available focal intervention strategy. through medical records) in 13 of 17 (76%) patients. Among OCT imaging studies of retinal laminar architecture with the detectable ERGs (8/13; 62%), b-wave amplitudes to a max- colocalized visual sensitivity measurements in patients with

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FIGURE 3. Details of the retinal laminar structure in MYO7A-USH1B examined by FD-OCT. (A) Cross-sectional OCT along the horizontal meridian from the fovea extending into the temporal retina in a normal subject and a patient (F5,P2) with MYO7A-USH1B. Arrows: INL, OPL, ONL, and OLM layers in both scans. Inset: scan location on a schematic of the left retina. Calibration bar at right. (B) INL, OPL, and ONL thickness measure- ments along the horizontal meridian into temporal retina in the patient (circles) compared with normal limits (shaded gray areas: mean Ϯ 2 SD; INL and OPL, n ϭ 9, ages 8–48 years; ONL, n ϭ 25, ages 5–58 years). The change of symbol in INL measurements at ϳ8 mm temporal to the fovea (from circles to diamonds) represents change to a hyporeflective layer that is continuous with the INL (but not known to be composed only of INL cells). Bar beneath the ONL thickness graph defines OLM visibility (white) and abnormal prominence (hatched). Brackets above patient scan in (A) and graphs in (B) define locations of four contiguous subzones (a–d) of the transition between normal and severely abnormal retinal architecture.

MYO7A-USH1B led to our finding that large regions of retina standard for diagnosis and monitoring of RP.22 Indeed, full-field can be normal in structure and function. Such findings were ERGs in patients with MYO7A-USH1B are severely reduced in not anticipated because the traditional notion is that retinal amplitude,23 but plans to deliver a focal treatment demand degenerations are retina-wide diseases with various levels of understanding of the disease details in relatively small retinal severity, a percept that derives from full-field ERGs, the gold regions.

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FIGURE 4. Subzones of the transition from normal to abnormal retina in MYO7A-USH1B. (A) Cross- sectional OCT scans in four representative patients with MYO7A-USH1B. Scans are extending from the fovea into the temporal, superior, or inferior retina. Insets: locations of each scan. White rectangles: limits of each of transition zone subdivisions (labeled a–d). (B) INL, OPL, and ONL thickness measurements in representative examples of the four subdivisions of the transition zone. OLM visibility is marked as hatched bar beneath the ONL thickness graph of subzone a. Insets: location of the subzone; shaded areas: normal limits (mean Ϯ 2 SD).

Adjacent to normal retina in many patients was a readily The defining histopathologic feature of inherited retinal identifiable transition zone to abnormal retina, and there were degenerations, whether in postmortem human donor tissue or stereotypical anatomic features within this zone. The assump- in animal models, is loss of photoreceptors. Traditionally, quan- tion is that these features are markers of a continuum of change titation has occurred by ONL cell counting or ONL thickness that leads from normal retinal architecture with normal func- measurements.28–30 In vivo micron-level measurements of tion to remodeled retina with little or no function. Among the ONL thickness in human retinal degenerations were not pos- more subtle OCT signs of abnormality was increased promi- sible until the advent of OCT. Quantitative comparisons of nence of the OLM. The OLM represents the most distal pro- frozen sections of normal and abnormal animal retina to OCT cesses of Mu¨ller glia, which interconnect with neighboring images of the same retinas indicated that OCT lamination was Mu¨ller cells and photoreceptors by specialized junctions.24 plausible to relate to retinal histopathology.10,31 OCT has now Increased OLM visibility on OCT is likely a marker of altered become nearly routine as a method to understand retinal de- Mu¨ller cell properties, such as swelling of microvilli or hyper- generative diseases and disease mechanisms,11,13,16,19,32–35 reactivity, in response to local photoreceptor cell stress or and more recently, OCT has been used to identify appropriate death. Of interest, endothelin receptor B immunoreactivity, a candidates for therapeutic initiatives.12,14,36 Higher resolution Mu¨ller cell response to photoreceptor injury or disease, was imaging in the present study led to the observation that there reported to be especially prominent in the OLM after light is a constellation of laminar changes that accompany reduction exposure to the rodent retina.25 Further, it has been postulated in ONL thickness in the MYO7A-USH1B retina. There was that the OLM is the site of interconnection between the Usher abnormal thickening of the OPL and INL in what we termed protein network and the proteins forming the Crumbs com- subzone b. The OPL is the site of photoreceptor synapses and plex.26 How such interactions lead to this optical–structural these are enveloped by Mu¨ller cell processes24; the INL is a abnormality is not known. The increase in OLM visibility is in layer with many retinal neurons and Mu¨ller cell nuclei. A contrast to morphologic evidence of OLM disruption in more parsimonious explanation is that both OPL and INL thickening advanced stages of retinal degeneration.27 are phenomena related to Mu¨ller cell hypertrophy or hyperre-

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FIGURE 5. Visual function and retinal structure in the transition zone of patients with MYO7A-USH1B. (A, B) Light-adapted sensitivity profiles to a white stimulus across the central retina in the horizontal (A) and vertical (B) meridians in patients with MYO7A-USH1B (n ϭ 7, ages 19–49 years). Shaded area: normal range (mean Ϯ 2 SD; n ϭ 9; ages 19–48 years). (C) Cross-sectional OCT image along the horizontal meridian from the fovea into the temporal retina of a 17-year-old patient with MYO7A-USH1B (F4,P1). Arrow: ONL. Bar above the cross-section indicates colocalized light-adapted sensitivities. Sensitivity scale to the left; dashed line: normal limit (mean Ϫ 2 SD). (D) ONL thickness expressed as the logarithm of the fraction of normal mean value for each measured location (in 0.6-mm bins) along the horizontal and vertical meridians, plotted as a function of colocalized visual sensitivity loss in representative patients with MYO7A-USH1B (n ϭ 7; ages 19–35 years). A linear regression is shown (gray line) with the 95% prediction interval (dashed lines).

activity in response to the photoreceptor apoptosis that ac- retain normal central function and structure. That leaves sub- counts for ONL thinning. In subzone b, the OLM varies from zones b and c as candidate regions for treatment. Assuming visible to not detectable. ONL thinning has been noted his- that photoreceptor apoptosis is the provocative event leading topathologically to be associated with a disappearance of the to OLM, OPL, and INL changes in these two subzones, an OLM.28 alteration in the pattern of laminar architecture may be a Subzone c shows further ONL thinning, a return of OPL to structural parameter suggesting efficacy. For example, there normal thickness, and greater increases of INL thickness. The would be no expectation of ONL increase but a decrease in underlying histopathology would most likely be major photo- OPL and INL thickness could be an indirect sign of less pho- receptor loss with possible rod neurite outgrowth,28 distur- toreceptor stress or loss. From an optimistic viewpoint, the bance of the integrity of the OPL, and eventual intermingling of region could also show a difference in relationship between the two nuclear layers.13,19 Subzone d, a late-stage OCT appear- structure and function; outer segment material could increase ance that we have documented in many other retinal degenera- as photoreceptor ciliary function improves, and sensitivity tions with lesser resolution OCT instruments,13,15,16,32–35 is gains would follow. Our finding of similar subzone patterns in characterized by a loss of normal laminar architecture. Under- other retinal meridians in the same individual should be fol- lying histopathology is speculated to be nearly complete pho- lowed up by formal interocular comparisons. A potentially toreceptor loss, a residual cell layer populated by hypertrophic expeditious method of evaluating a focal therapy may be to Mu¨ller glial cell nuclei and residual INL cells almost approxi- compare the properties of structure and function in the region mate to RPE cells, and a thick vitread layer of IPL processes, of the injection to noninjected sites that show similar proper- ganglion cells, nerve fibers, and epiretinal membrane from ties on baseline examinations. Mu¨ller glial response. These laminar abnormalities are likely to represent more longstanding disease and extend into the pe- riphery where there may be another transition zone to less Acknowledgments 28,37 advanced retinopathy. The authors thank Elaine Smilko, Leigh Tolley, and Waldo Herrera for What is the value of defining and then subdividing the critical help. transition from normal to abnormal retina? In focal treatment strategies, such as subretinal gene replacement, there is a need to know where exactly in the retina it is most sensible and safe References to perform the injection of vector-gene. Gliotic and remodeled 1. Williams DS. Usher syndrome: animal models, retinal function of retina representing late stages of retinal degeneration (subzone Usher proteins, and prospects for gene therapy. Vision Res. 2008; d) could be valuable as sites for the retinotomy, but not sites 48(3):433–441. for the entire injection volume. Subfoveal injections may lead 2. Maerker T, van Wijk E, Overlack N, et al. A novel Usher protein to greater risk than potential benefit38,39 in those patients who network at the periciliary reloading point between molecular

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