Residual Function in RPE65 Deficiency: Cones, Rods, or Both
ERGs were abnormal in all patients with RPE65-LCA tested. There were no detectable waveforms in 41% of the patients tested, and the other 59% had signals to only some stimuli (described later). Representative ERGs in RPE65-LCA were compared with normal waveforms and those from a disease with primary rod dysfunction, autosomal dominant retinitis pigmentosa (adRP) caused by rhodopsin (RHO) gene mutation (). The normal ERG to a dim blue light flash in the dark-adapted state represents a rod-mediated b-wave. A white light flash, dark-adapted, elicits a faster and larger waveform with both rod and cone (i.e., mixed) contributions, but is rod dominated. Cone ERGs are recordable in the light-adapted state to single flashes of light or to flickering light at 29 Hz. Severe early-onset rod disease and residual (but abnormal) cone function is exemplified by an 8-year-old patient with adRP who had the R135L RHO mutation. There was no measurable rod b-wave and, unlike the normal, waveforms elicited with white light stimuli in the dark- and light-adapted states were similar in shape and magnitude. There was a retained but abnormal flicker signal.
FIGURE 1 Measures of residual function in RPE65-LCA. (A) Standard ERGs in patients with RPE65-LCA. Rod, mixed cone-rod, and cone ERGs from six representative patients with RPE65-LCA (age range, 5–23 years) were compared with ERGs of a patient with a rhodopsin (more ...)
None of the patients with RPE65-LCA showed ERG responses to the dim blue light stimuli that normally evoke rod b-waves (). Twelve patients (P1, P2, P13, P16, P19, P22, P23, P25–P28, and P30) had no detectable ERGs to any stimuli; eight patients (P5, P7, P8, P11, P15, P18, P21, and P24) had only small amplitude (range, 2–4 µV) flicker ERGs; and nine patients (P3, P4, P6, P9, P10, P12, P14, P17, and P20) showed similar responses to dark- and light-adapted white stimuli and flicker waveforms with amplitudes ranging from 4 to 35 µV (normal mean ± SD = 172 ± 35 µV). We also used bright flashes to elicit ERGs in four patients (P5, P17, P18, and P22); there were no detectable responses (data not shown). In summary, the detectable ERGs in RPE65-LCA showed the pattern of a retinopathy with such severe rod dysfunction that it is not detectable with this technique; residual and abnormal cone function were measurable in many of the patients.
FST was performed in 22 patients with RPE65-LCA by using white and chromatic stimuli (). Sensitivity to the white stimulus in the patients with RPE65-LCA was abnormally reduced and showed a range of results representing sensitivity losses of 34 to 69 dB (, left). Chromatic measurements showed that more than half of the patients (13/22; 59%; age range, 6–27 years; mean age, 16) had rod mediation of the blue stimulus, indicating that there was severely reduced but detectable residual rod function. The remaining patients (9/22; 41%; age range, 5–46 years; mean age, 24) detected both red and blue stimuli with cone-mediated vision (, right). It can be concluded that residual rod function (and not only cone function) is detectable by psychophysics in patients with RPE65-LCA. There was no clear relationship of patient age to presence of residual rod function.
Localization of Residual Vision in the Visual Field
Kinetic visual fields with V-4e, a large bright target, were measurable in 29 of 30 patients. None of the patients was able to detect I-4e, the small bright target. A normal extent of kinetic field (defined as ≥90% for V-4e27
) was present in four patients (P8, P12, P14, and P20) at ages 10, 12, 19, and 23, respectively; there were no absolute scotomas detectable within these fields. The kinetic field of P20 at age 23 (, top left) exemplifies this pattern. Other patients (n
= 13; age range, 4–33 years) showed sizeable fields that were generally constricted and had reduced extent (39%–88% of normal). Such patterns are exemplified by P3 at age 6 (, top center). Later stage patterns showed either a residual central island, with or without peripheral islands, or only a far peripheral island (, right: P24, age 29; P27, age 41; P23, age 28). The graph of these data indicates a wide variation in the extent of kinetic field in the first two decades of life. Only islands of vision of limited extent were measurable after the third decade of life.
FIGURE 2 Kinetic perimetry in RPE65-LCA. (A) Kinetic field extent expressed as a percentage of normal and plotted as a function of age of patient. Longitudinal data are connected by lines. Representative kinetic fields illustrating early and later stage patterns (more ...)
Limited longitudinal data for kinetic fields are also plotted () and illustrated (). P8, at age 10 had a full kinetic field (to the V-4e target), but over the ensuing 11 years showed progressive loss of superior and then mid-peripheral fields; at age 21 there was a central island isolated by an absolute scotoma from a temporal peripheral island (). P5, at age 10, had generalized constriction of field (39% of normal) and a follow-up at age 17 years revealed only a small central island (). P18, at age 21, showed generalized constriction (38% of normal); at age 24, there was a residual central island with a temporal peripheral island ().
Static threshold perimetry in the dark-adapted state was performed in 17 of the patients. The highest intensity of the white stimulus used in the static perimetry is 1 log unit higher than that in the kinetic perimeter. Maps of dark-adapted sensitivity are shown for normal subjects (mean map of four individuals; age range, 22–61 years) compared with results from eight representative patients with RPE65-LCA. Patients are ordered (left to right in each row) by their mean sensitivity across the field. P14 and P20 had the highest mean sensitivities among the eight patients shown; however, these dark-adapted sensitivities were still approximately 5 log units reduced compared with those of normal subjects (mean normal average, 72 dB [SD, 1.6] versus 23 and 19.1 dB, respectively, for P14 and P20). The sensitivity map for P20 shows some decrement in peripheral sensitivity with relative central and mid-peripheral field preservation. Other dark-adapted sensitivity maps (P11, P8, and P17) show losses in the mid-peripheral field but with residual islands of central and peripheral function. More advanced stages (P21, P13, and P23) show extensive mid-peripheral scotomas separating central from peripheral vision. It is of interest that patients at advanced disease stages with only central islands by kinetic perimetry (n = 4) all showed detectable peripheral islands by dark-adapted static perimetry. The peripheral islands were equal or lower in sensitivity than the central islands. A patient with retained peripheral function but no measurable central function by kinetic perimetry (P23), had measurable central and peripheral islands with dark-adapted static perimetry and there was greater sensitivity in the periphery. Such findings are most likely attributable to the 1-log-unit brighter stimulus available with the static perimeter.
Dark-adapted perimetry results were summarized by plotting sensitivity as a function of eccentricity, and these cross-sectional data were grouped to arrive at a hypothetical severity sequence of residual visual loss (). These data support the notion that widespread loss of sensitivity occurs early in the disease and may progress to patchy loss, which then increases in severity. In normal subjects, sensitivity minimum is at fixation; there is an increase to a peak at ~12°; and a gradual decline occurs at greater eccentricities. Sensitivity in patients with RPE65-LCA was abnormally reduced at fixation but was still higher than at greater eccentricities, which were at least 4 log units reduced. The different patterns of loss at eccentricities >10° suggest a sequence of severity. At early stages (data from four patients, , top right), there were relatively homogeneous losses between ~10° and the periphery. Later stages showed prominent losses between 30° and 60° eccentricity, and these regional mid-peripheral losses deepened while function nearer fixation declined less dramatically (data from five patients, , lower left). The pattern at even later stages (data from four patients, , lower right) suggested that the 30° to 60° region of loss had become an absolute scotoma; the extent of this scotoma was greater than at the previous stage and involved more central field. Data from four of these patients of comparable age (19–23 years) were used to illustrate further the hypothesized sequence of residual vision loss in RPE65-LCA (). Relatively homogenous loss (P14) was followed by prominent mid-peripheral loss (P8) that can result in an absolute mid-peripheral scotoma (P21); finally, there was centripetal movement of the defect boundary and only central and/or peripheral islands of vision remained (P13).
FIGURE 3 Dark-adapted static threshold perimetry to a white stimulus in RPE65-LCA. (A) Sensitivities at 71 loci in normal subjects (N) and patients with RPE65-LCA are mapped to a pseudocolor scale representing 7 log units. Eight patient sensitivity maps are shown (more ...)
Relation of Residual Vision to Residual Photoreceptor Structure
Visual function and retinal structure in RPE65
-LCA are related in a complex manner because of dual defects that can affect vision in this disease: a biochemical blockade of the visual cycle and a complicating retinal degenerative process leading to photoreceptor cell loss.9,18,24
We asked the practical question of whether detectable dark-adapted sensitivity to the white stimulus of the static perimeter had any relationship to the presence of measurable photoreceptor layer (outer nuclear layer, ONL). The answer to this question could be of value in screening candidate patients for therapy and in directing local therapy to appropriate retinal regions, especially those regions in the peripheral retina where large deviations from the optical axis result in loss of signal on OCT scans due to optical distortions.
Vertical cross-sectional OCT images of the retina spanning approximately 8 mm to either side of the fovea were analyzed in 20 patients with RPE65-LCA for ONL thickness (). Vertical profiles of dark-adapted sensitivity were used to relate the presence or absence of visual sensitivity to ONL across this same region. Like the normal subject, P10 at age 12 had detectable visual sensitivity across the vertical meridian. There was a wide extent of measurable ONL (highlighted in blue on scans), although it was substantially reduced in thickness compared with normal. P22 at age 27, had ONL that extended from the fovea to approximately 3 to 4 mm superior and inferior; discontinuous with this ONL was a thinned segment of ONL detectable superiorly. Visual sensitivity was present in most of the region that showed detectable ONL.
FIGURE 4 Relationship between dark-adapted visual function and retinal structure in RPE65-LCA. (A) Vertical cross-sectional retinal images using OCT compared with the presence or absence of visual response in corresponding locations. A 48-year-old normal subject (more ...)
To summarize the data in the 20 patients with RPE65-LCA, we sampled 487 loci along the vertical meridian for ONL thickness and detectable dark-adapted visual sensitivity (). There were 95 loci with no measurable vision; 71 of these loci (75%) also had no measurable ONL. The remaining 24 loci with no vision had <135 µm of ONL thickness (mean, 8 µm [SD, 3.5]). Among the 392 loci with detectable dark-adapted visual sensitivity, 377 (96%) loci showed measurable ONL (mean, 21 µm [SD, 15]). The conclusion from these data representing a wide central retinal region is that presence of dark-adapted visual sensitivity is highly likely to be associated with the presence of underlying ONL.