July 2016
Volume 57, Issue 9
Open Access
Articles  |   July 2016
Outer Retinal Changes Including the Ellipsoid Zone Band in Usher Syndrome 1B due to MYO7A Mutations
Author Affiliations & Notes
  • Alexander Sumaroka
    Scheie Eye Institute Department of Ophthalmology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States
  • Rodrigo Matsui
    Scheie Eye Institute Department of Ophthalmology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States
  • Artur V. Cideciyan
    Scheie Eye Institute Department of Ophthalmology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States
  • David B. McGuigan, III
    Scheie Eye Institute Department of Ophthalmology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States
  • Rebecca Sheplock
    Scheie Eye Institute Department of Ophthalmology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States
  • Sharon B. Schwartz
    Scheie Eye Institute Department of Ophthalmology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States
  • Samuel G. Jacobson
    Scheie Eye Institute Department of Ophthalmology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States
  • Correspondence: Samuel G. Jacobson, Scheie Eye Institute, University of Pennsylvania, 51 N. 39th Street, Philadelphia, PA 19104, USA; jacobsos@mail.med.upenn.edu
Investigative Ophthalmology & Visual Science July 2016, Vol.57, OCT253-OCT261. doi:10.1167/iovs.15-18860
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      Alexander Sumaroka, Rodrigo Matsui, Artur V. Cideciyan, David B. McGuigan, Rebecca Sheplock, Sharon B. Schwartz, Samuel G. Jacobson; Outer Retinal Changes Including the Ellipsoid Zone Band in Usher Syndrome 1B due to MYO7A Mutations. Invest. Ophthalmol. Vis. Sci. 2016;57(9):OCT253-OCT261. doi: 10.1167/iovs.15-18860.

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

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Abstract

Purpose: To study transition zones from normal to abnormal retina in Usher syndrome IB (USH1B) caused by myosin 7A (MYO7A) mutations.

Methods: Optical coherence tomography (OCT) scattering layers in outer retina were segmented in patients (n = 16, ages 2–42; eight patients had serial data, average interval 4.5 years) to quantify outer nuclear layer (ONL) and outer segments (OS) as well as the locus of EZ (ellipsoid zone) edge and its extent from the fovea. Static perimetry was measured under dark-adapted (DA) and light-adapted (LA) conditions.

Results: Ellipsoid zone edge in USH1B-MYO7A could be located up to 23° from the fovea. Ellipsoid zone extent constricted at a rate of 0.51°/year with slower rates at smaller eccentricities. A well-defined EZ line could be associated with normal or abnormal ONL and/or OS thickness; detectable ONL extended well beyond EZ edge. At the EZ edge, the local slope of LA sensitivity loss was 2.6 (±1.7) dB/deg for central transition zones. At greater eccentricities, the local slope of cone sensitivity loss was shallower (1.1 ± 0.4 dB/deg for LA) than that of rod sensitivity loss (2.8 ± 1.2 dB/deg for DA).

Conclusions: In USH1B-MYO7A, constriction rate of EZ extent depends on the initial eccentricity of the transition. Ellipsoid zone edges in the macula correspond to large local changes in cone vision, but extramacular EZ edges show more pronounced losses on rod-based vision tests. It is advisable to use not only the EZ line but also other structural and functional parameters for estimating natural history of disease and possible therapeutic effects in future clinical trials of USH1B-MYO7A.

Advances in studying and monitoring human inherited retinal degenerations (IRDs) have occurred as a result of the use of optical coherence tomography (OCT). Traditional en face viewing of the ocular fundus has now been supplemented by cross-sectional images that provide understanding of the retinal lamination previously reserved for rare histopathology from postmortem eye donor tissue.1 Optical coherence tomography resolution has improved and sufficient studies comparing retinal tissue to OCT structure have been performed to add greater certainty about interpretations of how the laminar architecture relates to human retinal substructure.25 Although OCT will continue to advance, the current technology can be used to assay retinal structure in IRDs, changes with disease progression, and response to novel interventions.68 
Very recently, evidence has been put forth that a specific OCT feature of the photoreceptor laminae, the inner/outer segment border or ellipsoid zone (EZ), is able to be used to reliably determine the natural history of photoreceptor progression in X-linked retinitis pigmentosa (XLRP).9,10 This is a major step forward, considering the need for such parameters in current and future clinical trials of IRDs. Beyond natural histories of IRDs, there is the hope that such objective markers will also be potential outcome measures and permit decisions over a relatively short term about safety and efficacy of therapies. The road is now paved for such measurements to be made by individual investigators or in multicenter treatment trials by reading centers, for example, that can receive OCT scans and quantify this parameter. 
Previous studies that demonstrate measurements of EZ extent have focused on the central retina.9,11,12 Many future trials, however, are not being designed only to treat the central retina in which the EZ extent may be readily measurable. If there is intent to evaluate and treat more than the central retina or even to exclude treatment of the central retina, does EZ extent remain the marker to quantify, independent of retinal location and disease type? We have previously studied the retinal laminar architecture in the multisensory disorder with hearing and visual losses known as Usher syndrome.1315 Considering progress toward clinical trials in Usher syndrome type 1B (USH1B) due to MYO7A mutations,16,17 we chose to ask questions about the EZ extent, as well as its relationship to other outer retinal parameters and to vision in a cohort of patients with this disorder. 
Methods
Human Subjects
Sixteen patients (ages 2–42 years) with USH1B and known MYO7A mutations were included (Table). Patients had a complete eye examination. A group of normal subjects (n = 28, ages 5–58 years) were also studied. Informed consent was obtained; procedures followed the Declaration of Helsinki, and there was institutional review board approval. 
Table.
 
Characteristics of the MYO7A Usher1B Patients
Table.
 
Characteristics of the MYO7A Usher1B Patients
Optical Coherence Tomography
Optical coherence tomography was performed mainly with a spectral-domain (SD) OCT system (RTVue-100; Optovue, Inc., Fremont, CA, USA). Some patients were studied with time-domain OCT (OCT1 and OCT3; Carl Zeiss Meditec, Dublin, CA, USA). Our recording and analysis techniques have been published.2,18,19 In brief, the OCT protocol included 4.5- or 9-mm line scans along horizontal and vertical meridians crossing the fovea and extending to 9 mm into the periphery. Each scan was repeated three or more times. Postacquisition processing of OCT data was performed with custom programs (MatLab 7.5; MathWorks, Natick, MA, USA). Lateral sampling density of SD-OCT high-density scans (HD line) composed of 4091 A-scans was reduced by averaging neighboring A-scans within 8 μm to increase the signal-to-noise ratio. Scans were aligned by straightening the major RPE reflection and overlapping scans digitally merged to cover up to 30° in each direction. Repeated time-domain OCT scans were aligned and averaged to get scans with higher signal-to-noise ratio. Quantitation of retinal layers was performed manually as a function of eccentricity every 0.5° using published methods.20 The hyposcattering outer nuclear layer (ONL) was defined between the hyperscattering outer plexiform layer (OPL) and the hyperscattering outer limiting membrane (OLM) and included the anatomic layers of both ONL and Henle fiber layer (HFL). The outer segment (OS) layer thickness was measured between the EZ line and the hyperscattering peak near the interface of OS tips and apical RPE processes.21 In the current work we will use the term “EZ extent” to refer to the distance between the fovea and the location “EZ edge” where the EZ band becomes indistinguishable from the RPE along both the horizontal and vertical meridians. Previously used “EZ width”9 would be approximately twice the EZ extent used here assuming symmetry around the fovea. Scans with an indistinguishable EZ band were excluded. In patients with longitudinal data, annual EZ constriction rate along each meridian was estimated by dividing the difference in EZ extents at first and last visits by the time difference. Previously used annual “EZ loss” rate9 would be approximately twice the annual EZ constriction rate presented here. 
Visual Thresholds
Static perimetry was performed with a modified computerized perimeter (HFA-750i analyzer; Zeiss-Humphrey, Dublin, CA, USA). The stimuli (200-ms duration, 1.7°-diameter target) were spaced at 2° intervals along the horizontal and vertical meridians; the goal was to colocalize these data with the OCT scans. Both scotopic and photopic sensitivities were measured.22,23 Scotopic sensitivities used 500- and 650-nm stimuli; the sensitivity difference between the two stimuli allowed for determination of photoreceptor mediation.22,23 For rod-mediated loci, rod function was taken as the sensitivity to the 500-nm stimulus. Photopic sensitivity was measured with a white light stimulus on a white background (10 cd/m2). 
The report of a difference in visual sensitivity between loci inside versus outside the EZ edge in XLRP10 prompted us to determine whether such a trend was present in the data of USH1B. The previously published method used sensitivity data from loci acquired with a Humphrey 30-2 grid10 that used 6° spacing and did not colocalize to the retinal locations scanned with the OCT. Our sensitivity results, on the other hand, were from profiles spaced at an interval of 2° colocalized with the OCT scans. Three retinal directions (temporal, superior, and inferior) were used to provide sensitivities “just inside” and “just outside” EZ edge. For comparability with the published work, “just inside” was defined as the average of sensitivities at 2° and 4° from EZ edge toward the inside (less degenerate) direction; “just outside” was defined as the average of sensitivities at 2° and 4° from EZ edge toward the outside (more degenerate) direction. Further to these averages, we also evaluated profiles (at 2° spacing) on either side of the EZ edge to provide finer resolution sensitivity loss data. Such data compensate for the normal reduction of raw sensitivity values when comparing more central locations with more peripheral locations. 
Results
EZ Measurements in the Retina of USH1B Patients
The EZ extent was measured in serial OCT data from USH1B patients with definable EZ bands to determine if structural changes were detectable between visits. The time intervals between visits ranged from 1.3 to 8.3 years (average, 4.5 years). Representative horizontal and vertical cross sections are shown for USH1B patient (P)7 at ages 15 and 17 years (Fig. 1A). The EZ band is highlighted (orange), and the RPE inner boundary (between the OS and the RPE) is also shown (dark red line). 
Figure 1
 
Ellipsoid zone extent and the annual rate of EZ constriction in different retinal regions of USH1B. (A) Optical coherence tomography scans along horizontal (left) and vertical (right) meridians through the fovea for P7 at ages 15 (top) and 17 (bottom). The EZ line is highlighted in orange and the RPE inner boundary in dark red. Ellipsoid zone extent is measured along temporal (EZT), superior (EZS), and inferior (EZI) meridians. Icons: locations of cross-sectional scans. (B) Ellipsoid zone extent versus age for eight USH1B patients (ages 5–34 at first visit) along the three meridians; longitudinal data are connected by lines. (C) Average of initial EZ extents (orange bars, left axis) and the average rate of EZ constriction (gray bars, right) for the three retinal regions. (D) Individual rates of EZ constriction as a function of initial EZ extent. Black line is the regression line.
Figure 1
 
Ellipsoid zone extent and the annual rate of EZ constriction in different retinal regions of USH1B. (A) Optical coherence tomography scans along horizontal (left) and vertical (right) meridians through the fovea for P7 at ages 15 (top) and 17 (bottom). The EZ line is highlighted in orange and the RPE inner boundary in dark red. Ellipsoid zone extent is measured along temporal (EZT), superior (EZS), and inferior (EZI) meridians. Icons: locations of cross-sectional scans. (B) Ellipsoid zone extent versus age for eight USH1B patients (ages 5–34 at first visit) along the three meridians; longitudinal data are connected by lines. (C) Average of initial EZ extents (orange bars, left axis) and the average rate of EZ constriction (gray bars, right) for the three retinal regions. (D) Individual rates of EZ constriction as a function of initial EZ extent. Black line is the regression line.
The EZ extent from the fovea was measured in temporal, superior, and inferior directions considering that asymmetries of outer retinal structure, especially in the superior–inferior direction, have been previously observed at different disease stages in USH1B.14,15 Results of measurements plotted against age at visit are shown for eight patients (Fig. 1B). At first visits, EZ extent was 7.7° (range, 1.8°–17.4°), whereas at last visits, EZ extent was significantly (Wilcoxon signed rank test, P < 0.001) lower at 5.7° (1.2°–15.2°). The relationship between EZ extent and age appeared curvilinear by inspection. However, an exponential model with an invariant rate was not consistent with the data; there was a significant (P = 0.007) correlation between EZ rate and EZ extent. Thus a simpler linear model of progression was used for this limited data set until more information allows evaluation of models of greater complexity. Using the simple linear model, the annual rate of EZ constriction was calculated by dividing the difference between EZ extents at first and last visit by the time interval. The mean rate of EZ constriction in USH1B was 0.51°/year (0–5.2°/year), which was comparable to 0.43°/year (half of 0.86°/year9) previously reported in XLRP using a similar linear approach. Spatial distribution of the transition zones was not isotropic; the initial EZ extents at first visit differed along the three retinal meridians considered (1-way repeated measures ANOVA, P = 0.049). In the superior retina, the average extent was 9.6° (2.3°–17.4°). In the temporal retinal direction, the average extent was 8.6° (1.8°–16.3°), and for the inferior direction it was 5.2° (2.5°–11.8°) (Fig. 1C). Superior initial extent was significantly (P = 0.047) different than inferior, whereas superior versus temporal and temporal versus inferior extents were not significantly different (pairwise multiple comparisons, Student-Newman-Keuls method). The rates of EZ constriction showed progressive reduction of 0.98° (0–5.2), 0.4° (0–1.6), and 0.15 (0–0.49)°/year for superior, temporal, and inferior, respectively (Fig. 1C). Qualitatively, the data suggested that at early stages of disease, when the initial EZ extent is wider, there is faster progression. At later stages of disease, the EZ extent becomes limited and surrounds mainly the very central retina and shows a slower progression. Across all available data, this relation could be described by a linear function (P = 0.005) with a coefficient of 0.11°/year per degree (Fig. 1D). Such a tendency has also been previously demonstrated in XLRP.9 We also evaluated the progressive thinning of the ONL at the EZ edge at first visit. Not unexpectedly, the rate of ONL thinning at the eccentricity of the EZ edge at first visit was greater at further eccentricities than that closer to the fovea (5.6 μm/year for >10° vs. 1.1 μm/year for <10°). 
Different Outer Segment Abnormalities Within the EZ Extent
Optical coherence tomography scans along the vertical meridian are shown for two USH1B patients representing relatively early disease stages (Fig. 2A). The ONL, the EZ band, and rod OS (ROS) layer are highlighted. The foveal region was excluded from ROS segmentation up to an eccentricity of 2°, beyond which the majority of photoreceptors are rods in normal retinas.24 Patient 2 and P1, both at age 2 years, have scans that appear qualitatively normal or nearly normal in structure. 
Figure 2
 
Relation of EZ line to ONL and ROS in two young USH1B patients. (A) Optical coherence tomography scans along the vertical meridian through the fovea for two patients in the early stages of disease. Photoreceptor layers and EZ line are colored for visibility: ONL (dark blue), EZ line (orange), and ROS (light blue). Icon: location of the cross-sectional scans. (B) Outer nuclear layer (blue line) and (C) ROS (cyan line) thickness for the above cross-sectional scans are shown in relation to normal limits (shaded area). Ellipsoid zone line extent is represented by the orange bar in (B).
Figure 2
 
Relation of EZ line to ONL and ROS in two young USH1B patients. (A) Optical coherence tomography scans along the vertical meridian through the fovea for two patients in the early stages of disease. Photoreceptor layers and EZ line are colored for visibility: ONL (dark blue), EZ line (orange), and ROS (light blue). Icon: location of the cross-sectional scans. (B) Outer nuclear layer (blue line) and (C) ROS (cyan line) thickness for the above cross-sectional scans are shown in relation to normal limits (shaded area). Ellipsoid zone line extent is represented by the orange bar in (B).
Outer nuclear layer thickness, EZ extent, and ROS thickness across the vertical meridian for these patients are shown in relation to normal limits (Figs. 2B, 2C). The ONL of P2 is normal in thickness and the EZ line can be defined across the entire scanned region. Rod OS thickness, however, is borderline low and subnormal inferiorly starting from 11° eccentric to the fovea. The other 2-year-old USH1B patient, P1, shows normal ONL thickness and a detectable EZ line across most of the scan length. Rod OS thickness, however, is abnormally reduced at all eccentricities. 
These data from young USH1B patients illustrate that there can be different degrees of OS thickness abnormality within the region defined by EZ extent.25,26 Although EZ measurements have been considered to be a more sensitive marker of change than other outer retinal thickness parameters,12,13 it would seem valuable at this level of our understanding of disease and effects of therapy to know also the OS and ONL layer thicknesses within the EZ extent. Such measurements have already been useful to monitor safety of subretinal gene augmentation therapy,6 and an increase in OS layer thickness may be the only objective parameter that changes with certain forms of therapy. 
Revisiting the Transition Zone in USH1B
The “transition zone” was first studied in USH1B and subdivided based on changes in thickness across the region of the ONL, OPL, and INL (inner nuclear layer). The goal was to learn enough about the progressive degenerative disease to be able to recommend a retinal location that could be targeted by subretinal gene therapy.14 Subsequent studies of transition zones in other IRDs occurred, and attention shifted from the entire retina to mainly the outer retina25,26; this line of study subsequently led to the current level of interest in EZ extent as an objective marker of measurable disease progress.1012,25 
We revisited the transition zone in USH1B considering this new emphasis. Patient 9 shows a transition between normal and abnormal structure (at ∼4° eccentricity from the fovea) in the central retina (Fig. 3A, left), which is the most studied region to date.25,26 In this zone, ONL and ROS thicknesses decrease and the EZ line disappears. Quantitation of these three outer retinal parameters is shown for P11, P7, and P6. Patient 11 illustrates that EZ extent is still detectable beyond the eccentricity where ONL is already reduced below normal limits. Rod OS thickness is normal at the foveal side of the transition zone but becomes abnormally reduced within a short distance well before ONL thinning and EZ edge. Patient 7 has a small retinal distance of normal ONL and then a decline; EZ extent is beyond the extent of normal ONL. Rod OS thickness is abnormally reduced throughout the zone and declines within it. Patient 6 exemplifies the pattern of abnormal ONL thickness across the central retina, detectable EZ width, and even more limited ROS. All USH1B scans that represent this pattern of continuously declining ONL and ROS thickness in the transition zone are shown (Figs. 3C, 3D). 
Figure 3
 
Transition zones in USH1B. (A) Optical coherence tomography scans along the vertical meridian for two patients showing transition between normal and abnormal retina. A section of the scan is magnified (outlined in yellow, superior retina) and the EZ line (orange) and RPE inner boundary (dark red) are marked. Left: a centrally located transition zone (4° eccentricity). Right: a more peripherally located transition zone (23° eccentricity). (B) Quantitation of ONL and ROS thickness and EZ extent for representative examples of central (left) and more peripheral (right) transition zones. Plots display ONL (dark blue line, top) and ROS (light blue line, bottom) thickness, superior to the fovea, in relation to normal limits (shaded area). Ellipsoid zone line extent is indicated by orange bar (top). (C, D) Outer nuclear layer and ROS thickness along the horizontal and vertical meridians through the fovea for the entire cohort of USH1B patients, separated according to the central (left) (n = 8) or more peripheral (right) (n = 8) location of the transition zone. Ellipsoid zone line extent (not shown) would correspond to where ROS thickness becomes zero.
Figure 3
 
Transition zones in USH1B. (A) Optical coherence tomography scans along the vertical meridian for two patients showing transition between normal and abnormal retina. A section of the scan is magnified (outlined in yellow, superior retina) and the EZ line (orange) and RPE inner boundary (dark red) are marked. Left: a centrally located transition zone (4° eccentricity). Right: a more peripherally located transition zone (23° eccentricity). (B) Quantitation of ONL and ROS thickness and EZ extent for representative examples of central (left) and more peripheral (right) transition zones. Plots display ONL (dark blue line, top) and ROS (light blue line, bottom) thickness, superior to the fovea, in relation to normal limits (shaded area). Ellipsoid zone line extent is indicated by orange bar (top). (C, D) Outer nuclear layer and ROS thickness along the horizontal and vertical meridians through the fovea for the entire cohort of USH1B patients, separated according to the central (left) (n = 8) or more peripheral (right) (n = 8) location of the transition zone. Ellipsoid zone line extent (not shown) would correspond to where ROS thickness becomes zero.
A second pattern of results is illustrated in the data of P12 (Fig. 3A, right). The change from normal to abnormal (located at 23° superior to the fovea, which was the furthest EZ edge observed in the current cohort of patients) is less gradual and more of a steep-angled decline, representing a transition zone more frequently found outside the very central retina. Rod OS thickness in many scans declined below normal limits before ONL became abnormally reduced but then remained at a plateau before a further decline to a nondetectable level (Fig. 3B, right). This pattern was more evident in the superior and temporal retina (Figs. 3C, 3D, right). 
Relationship of Photoreceptor Structural Changes to Vision Across Transition Zones
The relationship reported between light-adapted (LA) visual sensitivity by static perimetry and outer retinal laminae by OCT has led to consideration of the EZ line measurement as an objective marker for monitoring the natural history and possible treatment trial efficacy of RP.14,25,27 The LA sensitivity results in P16 at age 42 are shown in the horizontal meridian from fixation into the temporal retina (excluding the complex region near the optic nerve head) and across the vertical meridian (Fig. 4A). Light-adapted vision appears to decline across the EZ edge, a pattern reminiscent of the changes in vision inside versus outside the EZ edge previously published in XLRP.10 Thus, we studied the EZ edge and vision in four USH1B patients with central islands using comparable methods. Exact replication of the published method was not possible because loci were tested with the Cen 30-2 pattern on a 6° grid offset from the OCT scans10 compared to our 2°-interval profiles colocalized with our OCT scans. However, 6°-grid-equivalent data could be generated from our higher-density data by using the average values at 2° and 4° inside the EZ edge and 2° and 4° outside. The result in USH1B with central islands was a sensitivity difference of 14.2 (±5.1) dB (Fig. 4B, left), which was somewhat larger than the 8.9 dB reported in XLRP.10 A portion of the sensitivity differences estimated by either method would be expected to originate from the normal hill of vision in these central retinal areas where there is a natural central-to-peripheral sensitivity gradient. Thus, we also evaluated the details of sensitivity loss in the region of the EZ edge from 4° inside to 4° outside (Fig. 4B, right). There was 2.7 (±3.5) dB of loss 2° inside the EZ edge and 12.9 (±7.5) dB of loss 2° outside the EZ edge, resulting in a local vision loss gradient of 2.6 (±1.7) dB/deg at the EZ edge. 
Figure 4
 
Structure and function comparisons in USH1B. (A) Light-adapted (LA) sensitivity profiles with white stimuli along the horizontal meridian from fovea (F) to temporal (T) retina and in the vertical meridian from inferior (I) to superior (S) retina shown with the position of the EZ line extent (orange bar) in P16, age 42. Thin orange lines extending from the EZ line indicate the EZ edge. Gray band represents normal LA sensitivity with lower limit defined as mean − 2SD. (B) Left: LA sensitivity changes from inside to outside EZ edge (from 4 USH1B patients with transition zones in the central retina) for temporal, inferior, and superior directions; gray circles connected by lines are the individual data; superimposed solid black squares connected by black line represent the average decrease in LA sensitivity. Right: LA sensitivity loss profiles ± 4° from EZ edge; individual data (gray open circles connected with gray lines) and an average profile (black squares connected by black line) are shown. (C) LA sensitivity profiles in horizontal and vertical meridians shown with position of EZ line (similar to [A]) for three representative USH1B patients (P12, P14, and P11) with transition zones that extend beyond the central retina. (D) Dark-adapted (DA) sensitivity profiles along with the position of the EZ edge (orange bar) for the same three patients as in (C). R, rod mediation of the loci determined by two-color dark-adapted perimetry. Light blue band represents normal DA sensitivity with lower limit defined as mean − 2SD. (E) Left: LA sensitivity changes from inside to outside EZ line (from five USH1B patients with transition zones extending beyond the central retina). Right: LA sensitivity loss profiles ± 4° from EZ end (similar to [B]) (F) Left: DA sensitivity changes from inside to outside EZ line (from the same five patients with transition zones extending beyond the central retina). Light gray lines are the individual data; superimposed blue line is the average from the five patients. Right: DA sensitivity loss profiles ± 4° from EZ edge; individual data (gray open circles connected with gray lines) and an average DA profile (blue circles and line). Also plotted for comparison are ONL and ROS relative thickness changes (gray lines are individual data, dark blue is average ONL data, dark cyan is ROS average data). Ellipsoid zone extent (orange bar along horizontal axis) and limits (thin orange line) as before.
Figure 4
 
Structure and function comparisons in USH1B. (A) Light-adapted (LA) sensitivity profiles with white stimuli along the horizontal meridian from fovea (F) to temporal (T) retina and in the vertical meridian from inferior (I) to superior (S) retina shown with the position of the EZ line extent (orange bar) in P16, age 42. Thin orange lines extending from the EZ line indicate the EZ edge. Gray band represents normal LA sensitivity with lower limit defined as mean − 2SD. (B) Left: LA sensitivity changes from inside to outside EZ edge (from 4 USH1B patients with transition zones in the central retina) for temporal, inferior, and superior directions; gray circles connected by lines are the individual data; superimposed solid black squares connected by black line represent the average decrease in LA sensitivity. Right: LA sensitivity loss profiles ± 4° from EZ edge; individual data (gray open circles connected with gray lines) and an average profile (black squares connected by black line) are shown. (C) LA sensitivity profiles in horizontal and vertical meridians shown with position of EZ line (similar to [A]) for three representative USH1B patients (P12, P14, and P11) with transition zones that extend beyond the central retina. (D) Dark-adapted (DA) sensitivity profiles along with the position of the EZ edge (orange bar) for the same three patients as in (C). R, rod mediation of the loci determined by two-color dark-adapted perimetry. Light blue band represents normal DA sensitivity with lower limit defined as mean − 2SD. (E) Left: LA sensitivity changes from inside to outside EZ line (from five USH1B patients with transition zones extending beyond the central retina). Right: LA sensitivity loss profiles ± 4° from EZ end (similar to [B]) (F) Left: DA sensitivity changes from inside to outside EZ line (from the same five patients with transition zones extending beyond the central retina). Light gray lines are the individual data; superimposed blue line is the average from the five patients. Right: DA sensitivity loss profiles ± 4° from EZ edge; individual data (gray open circles connected with gray lines) and an average DA profile (blue circles and line). Also plotted for comparison are ONL and ROS relative thickness changes (gray lines are individual data, dark blue is average ONL data, dark cyan is ROS average data). Ellipsoid zone extent (orange bar along horizontal axis) and limits (thin orange line) as before.
For USH1B patients with transition zones that extend beyond a central island, we asked whether the relationship between structure and function remained similar to those observed in central islands of USH1B or XLRP. Considering the increasing contribution to the retinal structure (with eccentricity) of rod photoreceptors, we studied both LA and dark-adapted (DA) sensitivities in the USH1B patients in relation to the EZ edge. For example, in three USH1B patients (P12, P14, and P11), the EZ extent is plotted along with colocalized LA visual sensitivities (Fig. 4C). The transition from normal to abnormal sensitivity appears to coincide with the EZ edge, except in P12 and P14 temporally, where the functional transition from normal to abnormal is further eccentric to the EZ edge. The magnitude of sensitivity change from inside to outside the EZ edge was determined, as in the analyses of the central island transition zones described above. The estimate of LA visual sensitivity difference between inside and outside the EZ line edge in 5 USH1B patients (P10–P14, ages 10–24; Table) in 6°-grid-equivalent analysis was 6.3 (±2.9) dB (Fig. 4E, left), which was substantially smaller than that observed in central transition zones. When we evaluated the details of sensitivity loss in the region of the EZ edge from 4° inside to 4° outside (Fig. 4E, right), there was 1.4 (±1.9) dB of loss 2° inside the EZ edge and 5.6 (±1.8) dB of loss 2° outside the EZ edge, resulting in a relatively shallow local vision loss gradient of 1.1 (±0.4) dB/deg at the EZ edge. 
Similar analyses were performed for DA sensitivities, specifically seeking the relationship between rod-mediated loci (based on two-color DA perimetry22) and rod-dominated outer retinal structure (Figs. 4D, 4F). In the same three representative patients, the transition from normal to abnormal rod sensitivity was compared with EZ edge: In the nasal and inferior fields, the functional transition coincided with the EZ edge. In the superior field, the EZ edge tended to be further eccentric than the transition from normal to abnormal rod sensitivity. In these regions, there was a difference of 14.9 (±4.5) dB between inside and outside of the EZ edge using the 6°-grid-equivalent analysis (Fig. 4F, left). Details of DA rod sensitivity losses across the EZ edge (Fig. 4F, right) showed 7.2 (±3.2) dB of loss 2° inside the EZ edge and 21.8 (±3.0) dB of loss 2° outside the EZ edge, resulting in a local rod vision loss gradient of 2.8 (±1.2) dB/deg at the EZ edge. Dark-adapted rod sensitivity losses compared favorably with the gradual changes of ONL thickness fraction versus the steep changes of ROS length fraction and EZ edge. 
Discussion
Standard clinical assessments, such as visual acuity and visual fields, have long been used to describe the disease and stage of IRDs, but it is a new experience for this field to have to consider designing and implementing ways to monitor therapies for these previously incurable retinopathies. Lists of test methods and parameters beyond the standard clinical measurements have been provided (see summaries published previously28,29). Knowing sufficient detail about the disease expression is key; fitting the available and appropriate test to the disease and treatment strategy has become a challenge. There has been a preference for objective test parameters versus subjective parameters. For example, this led to conflicting opinions that followed publication of results of nutritional supplementation in a long-term clinical trial of RP (submicrovolt ERGs versus perceptual evidence of efficacy30,31). 
Optical coherence tomography has emerged over the past two decades as a clinically available technology to visualize cross-sectional retinal structure and has the appeal of permitting objective monitoring of disease.32 Relatively rapid advances have occurred in speed and resolution of OCT instrumentation and understanding of the anatomic basis of the scans; machine algorithms to analyze features of the OCTs are available; and for clinical research and trials, there are now commercial groups with secure Web sites that will serve as reading centers and receive uploaded digital images and provide analyses. 
In the case of IRDs, the difficulty has been to determine if there is a change in any retinal or visual parameter that would be detectable over a relatively short time (e.g., 1–2 years) in otherwise very slowly progressive conditions. Recently, the EZ extent has been proposed as a useful monitor of progressive retinal (and visual) loss in XLRP.10 As a result, the letters “EZ” have become common parlance among contract research organizations (CROs) hired to manage projects, collect data, and link clinical trial lists with other CROs ready to measure and report EZ extent. 
The current work followed up our earlier study of USH1B that characterized the OCT disease effects across the transition zone between relatively normal and severely abnormal retina.14 Building on the recent advances in XLRP and the use of the EZ line to monitor progression, we asked questions about the EZ extent in USH1B. Some patients had only residual central retinal islands remaining, and in these patients there is general agreement with the method and results from XLRP.10 For patients with less severe disease and remaining extramacular structure, the analyses become more complex, and there are sufficiently different patterns of results not to allow generalizing from data representing central retinal transition zones. As mentioned in the study of XLRP patients,10 horizontal midline scans, although most commonly collected in the clinic, may be only part of the characterization of the diseased retina. When we measured the EZ extent in three directions from the fovea (temporal, superior, and inferior) longitudinally over an average of 4.5 years, there was a range of changes that appear related to the initial EZ extent. The positive association we found between the initial EZ extent and its rate of constriction in USH1B has been noted previously in XLRP.9 However, not all of the variability was explainable. Different rates of progression within the retina of an individual and between patients with the same or different retinopathies must be considered (and explored) when designing how to monitor patients in natural history studies or in treatment trials. This observation suggests that staging of disease (based on such parameters) would be valuable in order to include a relatively homogeneous group of patients with comparable progression rates; results could otherwise be difficult to interpret or lead to erroneous conclusions. 
By definition, there is no OS material at the retinal location where the EZ line is no longer measurable. Our demonstration of a difference between OS thickness within the EZ extent in two young USH1B patients, however, serves to remind that there are complexities in retinal structure (and likely function, if measurable) based on more than EZ extent. The two patterns of transition zones we found in our USH1B patients suggest that different OS lengths within the EZ extent occur in more than the two 2-year-old patients. Whether a disease natural history slowly reduces the OS length of photoreceptors or a future treatment can increase the OS length of residual photoreceptors would not be expected to be detectable as an EZ extent, but could be important data to understand progression or efficacy in a clinical trial. If OS length was not measured and visual function was, there could be the confusing combination of increased function but no change in structure if only the EZ extent was unchanged. There are arguments against psychophysical testing as being more variable and OS length measurements more difficult.9,11 Yet, at this early time in our attempts to estimate natural history of different IRDs and the possible therapeutic effects in clinical trials, we should seek not only simplicity of measurement but also the greatest understanding possible of altered mechanism by disease or intervention. 
Further, we examined visual changes that occur in concert with the structural changes. Whereas, standard LA static perimetry (traditionally designed for glaucoma monitoring but also found in retinal disease testing protocols) can be used to relate to structural parameters in residual central retina, which is assumed to be mainly cone mediated at late disease stages, it would seem less appropriate when the residual structure is likely to be rod dominated. The recently published method to determine the sensitivity inside and outside the EZ edge is attractive10; but use of a commonly available test paradigm (Cen 30-2) resulted in a lack of colocalization of OCT scans and sensitivity measurements. In subsequent studies seeking outcomes in IRDs with relatively stable fixation, it may be worthwhile to consider collecting data that are determined at similar retinal loci, enabling more direct comparisons. There is sufficient understanding of normal rod-to-cone ratios across the retina24 and disease effects in IRDs to estimate that the more extramacular retinal regions (such as illustrated with ONL and OS thickness data in Figs. 3C, 3D, right) are rod-dominated structure until the transition to loss of structure at greater eccentricities. Mismatching cone-based function with rod-dominated structure, although convenient for testing, is losing the potential to understand the effect of disease progression and possible therapeutic efficacy on rod photoreceptors. Automated DA static perimetry has been used in certain laboratories/clinics for more than 25 years22,23,3336 and even as outcomes in recent treatment trials.37,38 It would seem worthwhile to use such methods when assessing structure–function relationships across transition zones, at least in extramacular regions. When we used colocalized structure and function and also rod-mediated vision in comparisons of various parameters of structure in the more eccentric transition zones in USH1B, the data set was not the same as the LA results from the same loci (Figs. 4E, 4F). 
We plotted the sensitivity loss of rod-mediated loci along the transition zone at extramacular loci, and it was evident that ONL thickness was the most clearly related structural parameter to vision (Fig. 4F, right). In these regions, the critique that ONL can be contaminated by HFL reflections is less an issue.39,40 There is a well-studied relationship of structure to rod as well as cone function from many studies in a number of IRDs,19,25,37,41,42 and it would seem valuable to have such information to complement that from the EZ line extent. Also worth studying would be why there is measurable function eccentric to the end of the EZ line. This may be answered as resolution of the current imaging technology improves even further and there is detection of currently unresolvable details of residual outer retinal structure. Studies using fundus-viewing systems rather than free-viewing perimetry could also leave fewer questions about eye movement contamination of the data. 
Acknowledgments
Supported by grants from the The Chatlos Foundation, Foundation Fighting Blindness, Macula Vision Research Foundation, and Research to Prevent Blindness. 
Disclosure: A. Sumaroka, None; R. Matsui, None; A.V. Cideciyan, None; D.B. McGuigan III, None; R. Sheplock, None; S.B. Schwartz, None; S.G. Jacobson, None 
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Figure 1
 
Ellipsoid zone extent and the annual rate of EZ constriction in different retinal regions of USH1B. (A) Optical coherence tomography scans along horizontal (left) and vertical (right) meridians through the fovea for P7 at ages 15 (top) and 17 (bottom). The EZ line is highlighted in orange and the RPE inner boundary in dark red. Ellipsoid zone extent is measured along temporal (EZT), superior (EZS), and inferior (EZI) meridians. Icons: locations of cross-sectional scans. (B) Ellipsoid zone extent versus age for eight USH1B patients (ages 5–34 at first visit) along the three meridians; longitudinal data are connected by lines. (C) Average of initial EZ extents (orange bars, left axis) and the average rate of EZ constriction (gray bars, right) for the three retinal regions. (D) Individual rates of EZ constriction as a function of initial EZ extent. Black line is the regression line.
Figure 1
 
Ellipsoid zone extent and the annual rate of EZ constriction in different retinal regions of USH1B. (A) Optical coherence tomography scans along horizontal (left) and vertical (right) meridians through the fovea for P7 at ages 15 (top) and 17 (bottom). The EZ line is highlighted in orange and the RPE inner boundary in dark red. Ellipsoid zone extent is measured along temporal (EZT), superior (EZS), and inferior (EZI) meridians. Icons: locations of cross-sectional scans. (B) Ellipsoid zone extent versus age for eight USH1B patients (ages 5–34 at first visit) along the three meridians; longitudinal data are connected by lines. (C) Average of initial EZ extents (orange bars, left axis) and the average rate of EZ constriction (gray bars, right) for the three retinal regions. (D) Individual rates of EZ constriction as a function of initial EZ extent. Black line is the regression line.
Figure 2
 
Relation of EZ line to ONL and ROS in two young USH1B patients. (A) Optical coherence tomography scans along the vertical meridian through the fovea for two patients in the early stages of disease. Photoreceptor layers and EZ line are colored for visibility: ONL (dark blue), EZ line (orange), and ROS (light blue). Icon: location of the cross-sectional scans. (B) Outer nuclear layer (blue line) and (C) ROS (cyan line) thickness for the above cross-sectional scans are shown in relation to normal limits (shaded area). Ellipsoid zone line extent is represented by the orange bar in (B).
Figure 2
 
Relation of EZ line to ONL and ROS in two young USH1B patients. (A) Optical coherence tomography scans along the vertical meridian through the fovea for two patients in the early stages of disease. Photoreceptor layers and EZ line are colored for visibility: ONL (dark blue), EZ line (orange), and ROS (light blue). Icon: location of the cross-sectional scans. (B) Outer nuclear layer (blue line) and (C) ROS (cyan line) thickness for the above cross-sectional scans are shown in relation to normal limits (shaded area). Ellipsoid zone line extent is represented by the orange bar in (B).
Figure 3
 
Transition zones in USH1B. (A) Optical coherence tomography scans along the vertical meridian for two patients showing transition between normal and abnormal retina. A section of the scan is magnified (outlined in yellow, superior retina) and the EZ line (orange) and RPE inner boundary (dark red) are marked. Left: a centrally located transition zone (4° eccentricity). Right: a more peripherally located transition zone (23° eccentricity). (B) Quantitation of ONL and ROS thickness and EZ extent for representative examples of central (left) and more peripheral (right) transition zones. Plots display ONL (dark blue line, top) and ROS (light blue line, bottom) thickness, superior to the fovea, in relation to normal limits (shaded area). Ellipsoid zone line extent is indicated by orange bar (top). (C, D) Outer nuclear layer and ROS thickness along the horizontal and vertical meridians through the fovea for the entire cohort of USH1B patients, separated according to the central (left) (n = 8) or more peripheral (right) (n = 8) location of the transition zone. Ellipsoid zone line extent (not shown) would correspond to where ROS thickness becomes zero.
Figure 3
 
Transition zones in USH1B. (A) Optical coherence tomography scans along the vertical meridian for two patients showing transition between normal and abnormal retina. A section of the scan is magnified (outlined in yellow, superior retina) and the EZ line (orange) and RPE inner boundary (dark red) are marked. Left: a centrally located transition zone (4° eccentricity). Right: a more peripherally located transition zone (23° eccentricity). (B) Quantitation of ONL and ROS thickness and EZ extent for representative examples of central (left) and more peripheral (right) transition zones. Plots display ONL (dark blue line, top) and ROS (light blue line, bottom) thickness, superior to the fovea, in relation to normal limits (shaded area). Ellipsoid zone line extent is indicated by orange bar (top). (C, D) Outer nuclear layer and ROS thickness along the horizontal and vertical meridians through the fovea for the entire cohort of USH1B patients, separated according to the central (left) (n = 8) or more peripheral (right) (n = 8) location of the transition zone. Ellipsoid zone line extent (not shown) would correspond to where ROS thickness becomes zero.
Figure 4
 
Structure and function comparisons in USH1B. (A) Light-adapted (LA) sensitivity profiles with white stimuli along the horizontal meridian from fovea (F) to temporal (T) retina and in the vertical meridian from inferior (I) to superior (S) retina shown with the position of the EZ line extent (orange bar) in P16, age 42. Thin orange lines extending from the EZ line indicate the EZ edge. Gray band represents normal LA sensitivity with lower limit defined as mean − 2SD. (B) Left: LA sensitivity changes from inside to outside EZ edge (from 4 USH1B patients with transition zones in the central retina) for temporal, inferior, and superior directions; gray circles connected by lines are the individual data; superimposed solid black squares connected by black line represent the average decrease in LA sensitivity. Right: LA sensitivity loss profiles ± 4° from EZ edge; individual data (gray open circles connected with gray lines) and an average profile (black squares connected by black line) are shown. (C) LA sensitivity profiles in horizontal and vertical meridians shown with position of EZ line (similar to [A]) for three representative USH1B patients (P12, P14, and P11) with transition zones that extend beyond the central retina. (D) Dark-adapted (DA) sensitivity profiles along with the position of the EZ edge (orange bar) for the same three patients as in (C). R, rod mediation of the loci determined by two-color dark-adapted perimetry. Light blue band represents normal DA sensitivity with lower limit defined as mean − 2SD. (E) Left: LA sensitivity changes from inside to outside EZ line (from five USH1B patients with transition zones extending beyond the central retina). Right: LA sensitivity loss profiles ± 4° from EZ end (similar to [B]) (F) Left: DA sensitivity changes from inside to outside EZ line (from the same five patients with transition zones extending beyond the central retina). Light gray lines are the individual data; superimposed blue line is the average from the five patients. Right: DA sensitivity loss profiles ± 4° from EZ edge; individual data (gray open circles connected with gray lines) and an average DA profile (blue circles and line). Also plotted for comparison are ONL and ROS relative thickness changes (gray lines are individual data, dark blue is average ONL data, dark cyan is ROS average data). Ellipsoid zone extent (orange bar along horizontal axis) and limits (thin orange line) as before.
Figure 4
 
Structure and function comparisons in USH1B. (A) Light-adapted (LA) sensitivity profiles with white stimuli along the horizontal meridian from fovea (F) to temporal (T) retina and in the vertical meridian from inferior (I) to superior (S) retina shown with the position of the EZ line extent (orange bar) in P16, age 42. Thin orange lines extending from the EZ line indicate the EZ edge. Gray band represents normal LA sensitivity with lower limit defined as mean − 2SD. (B) Left: LA sensitivity changes from inside to outside EZ edge (from 4 USH1B patients with transition zones in the central retina) for temporal, inferior, and superior directions; gray circles connected by lines are the individual data; superimposed solid black squares connected by black line represent the average decrease in LA sensitivity. Right: LA sensitivity loss profiles ± 4° from EZ edge; individual data (gray open circles connected with gray lines) and an average profile (black squares connected by black line) are shown. (C) LA sensitivity profiles in horizontal and vertical meridians shown with position of EZ line (similar to [A]) for three representative USH1B patients (P12, P14, and P11) with transition zones that extend beyond the central retina. (D) Dark-adapted (DA) sensitivity profiles along with the position of the EZ edge (orange bar) for the same three patients as in (C). R, rod mediation of the loci determined by two-color dark-adapted perimetry. Light blue band represents normal DA sensitivity with lower limit defined as mean − 2SD. (E) Left: LA sensitivity changes from inside to outside EZ line (from five USH1B patients with transition zones extending beyond the central retina). Right: LA sensitivity loss profiles ± 4° from EZ end (similar to [B]) (F) Left: DA sensitivity changes from inside to outside EZ line (from the same five patients with transition zones extending beyond the central retina). Light gray lines are the individual data; superimposed blue line is the average from the five patients. Right: DA sensitivity loss profiles ± 4° from EZ edge; individual data (gray open circles connected with gray lines) and an average DA profile (blue circles and line). Also plotted for comparison are ONL and ROS relative thickness changes (gray lines are individual data, dark blue is average ONL data, dark cyan is ROS average data). Ellipsoid zone extent (orange bar along horizontal axis) and limits (thin orange line) as before.
Table.
 
Characteristics of the MYO7A Usher1B Patients
Table.
 
Characteristics of the MYO7A Usher1B Patients
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