RDS Gene Mutations Causing Retinitis ... - Semantic Scholar

RDS Gene Mutations Causing Retinitis Pigmentosa or Macular Degeneration Lead to the Same Abnormality in Photoreceptor Function Colin M. Kemp,* Samuel G. Jacobson* Artur V. Cideciyan* Alan E. Kimura,"\ Val C. Sheffield,^ and Edwin M. Stonef

Purpose. To investigate functional abnormalities in mutations in the peripherin (RDS) gene leading to different clinical types of autosomal dominant retinal disease—macular degeneration and retinitis pigmentosa. Methods. Patients from two families, one with a mutation in codon 167 (Glyl67Asp) leading to macular degeneration and another with a mutation in codon 210 (Pro210Ser) leading to retinitis pigmentosa, were studied with clinical examinations and measurements of rod and cone sensitivities and dark adaptation, electroretinography, and rhodopsin levels. Results. Mildly affected patients had sizable rod and cone electroretinograms, reduced levels of rhodopsin, and minor losses of sensitivity. In both mutations, there were delays of rod and cone dark adaptation after bleaching, and the adaptational abnormalities were observed in peripheral and central retinal locations. Analysis of the kinetics of rod adaptation indicates that the underlying abnormalities are similar in both mutations and that the effects of the mutations are similar to those caused by mild systemic vitamin A deficiency. Conclusions. Patients with the Glyl67Asp and Pro210Ser mutations in the peripherin/RDS gene have widely different clinical phenotypes but show the same abnormality, slowed dark adaptation, of rod and cone photoreceptor function. The similarities of the characteristics of the adaptational abnormalities in the two genotypes suggest that, in addition to the structural roles normally assumed for it, peripherin influences or participates in the function of the visual cycle. Invest Ophthalmol Vis Sci. 1994;35:3154-3162.

X he peripherin/RDS gene encodes a 346 amino acid glycoprotein found in both rod and cone photoreceptor outer segments. Peripherin is located within the disk membranes containing the visual pigment and is believed to play an important role in the maintenance of lamellar structure.1"3 Mutations in the peripherin/ RDS gene have been shown to lead to retinal degenerations, both in the mouse4"6 and in man.7"12 In man, From the *Department of Ophthalmology, University of Miami School of Medicine, liascom Palmer Eye Institute, Miami, Florida, and the ^Department of Ophthalmology, University of Iowa Hospitals and Clinics, Iowa City, Iowa. Supported in part by Public Health Service research grants EY05627 (SGJ) and EY08426 (EMS); the National Retinitis Pigmentosa Foundation, Baltimore, Maryland; the George Gund Foundation, Cleveland, Ohio, and the C. S. O'Brien Center for Macular Diseases and Research to Prevent Blindness, Inc., New York, New York. Dr. Stone is a Research to Prevent Blindness Dolly Green Scholar. Submitted for publication December 16, 1993; revised February 10, 1994; accepted February 15, 1994. Proprietary interest category: N. Reprint requests: Dr. Colin M. Kemp, Department of Ophthalmology, University of Miami School of Medicine, Bascom Palmer Eye Institute, 1638 N. W. 10th Avenue, Miami, FL 33136.

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the first clinical phenotype reported to result from peripherin/RDS mutations was retinitis pigmentosa (RP), in which typically there is night blindness and progressive loss of peripheral vision.78 Macular degenerations, diseases that affect mainly the central retina, were also found to be caused by mutations in this gene.10"11 Recently, both RP and macular degeneration were reported to occur in a family with a peripherin/RDS mutation.12 Why RDS mutations lead to such clinically different disease phenotypes is unknown. To increase our understanding of disease expression in patients with peripherin/RDS mutations, we used noninvasive techniques to measure rod and cone function in two families, one with RP caused by the proline-210-serine (Pro210Ser) peripherin/RDS mutation and the other with a macular degeneration caused by the glycine-167-aspartic acid (Glyl67Asp) mutation.10 We found that subjects with these very dif-

Investigative Ophthalmology & Visual Science, July 1994, Vol. 35, No. 8 Copyright © Association for Research in Vision and Ophthalmology

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Peripherin/RDS Mutations and Retinal Function

ferent clinical phenotypes had similar abnormalities in both rod and cone function, even in retinal regions where there were no manifest signs of disease. All patients had delays in rod and cone dark adaptation, and kinetic analysis of the rod data13"15 indicates that these abnormalities are not specific to the site of the mutation. Of interest, the abnormal characteristics are similar to those resulting from mild systemic vitamin A deficiency.1617 These results suggest that, as in vitamin A deficiency, function of the visual cycle may be impaired in these diseases resulting from peripherin/ RDS mutations. In addition to the structural roles usually assigned to it,1"3 therefore, peripherin may have a functional role in the photoreceptor outer segment. MATERIALS AND METHODS Subjects and DNA Analysis Patient MD1, a 56-year-old man, and patient MD2, his 41-year-old niece, were affected family members from an extensive pedigree with macular degeneration we had previously found was caused by a mutation in codon 167 of the peripherin/RDS gene.10 Patient RP1, a 42-year-old woman with symptoms and signs typical of RP, and patient RP2, her 15-year-old son, were from another family. Denaturing gradient gel electrophoresis of PCR amplified segments of the peripherin/RDS gene10 revealed a sequence change in exon 2 of these patients. DNA sequencing revealed this change to be a C to T transition in the first nucleotide of codon 210, resulting in the substitution of serine for proline at that position. The methods for DNA analyses have been described previously.10 Visual Function Tests Informed consent was obtained from the patients and from normal subjects involved in the study after the nature of the procedures had been explained fully. The research procedures were in accordance with institutional guidelines and the Declaration of Helsinki. Full-field electroretinograms (ERGs) were performed using previously described methods.1819 Rod ERGs were elicited with a dim blue flash (—0.1 log scot • td • s), dark adapted (normal mean b-wave amplitude = 299 /zV, SD = 52 /*V; mean implicit time = 76 msec, SD = 5 msec); a mixed cone and rod ERG with a bright white flash (5.4 cd s/m2), dark adapted (normal mean a-wave amplitude = 297 /xV, SD = 65 /xV; mean b-wave amplitude = 497 /iV, SD = 111 nV); cone ERGs at 1 Hz with white flashes (0.64 cd s/m2) on a white background light of 34 cd/m2 (normal mean bwave amplitude = 93 /iV, SD = 24 /xV; mean implicit time = 29 msec, SD = 1.5 msec); and cone flicker ERGs at 29 Hz with white flashes (0.64 cd s/m2) on a white background light of 6.9 cd/m2 (normal mean

peak-to-peak amplitude = 99 /xV, SD = 24 /xV; mean timing = 27 msec, SD = 1 msec). Goldmann kinetic perimetry was performed using targets V and I at intensity 4e (318 cd/m2) on a white background (10 cd/ m2). Static threshold perimetry was performed using 500-nm and 650-nm stimuli, dark adapted, and a 600 nm stimulus, light adapted, and a full-field test strategy with 75 loci on a 12° grid. Details of perimetric methods have been published.19'20 Dark adaptometry was tested with 500-nm and 650-nm stimuli. Baseline dark-adapted thresholds were determined after at least 3 hours of dark adaptation. For each patient, the recovery of sensitivity was measured after retinal exposures of 7.8, 6.9, and 6.3 log scot • td • s delivered with a yellow bleaching light (>520 nm), which are expected to bleach about 99%, 50%, and 15% of the original rhodopsin present, respectively. Further details of the methodology have been reported.21 The time courses of dark adaptation in the patients were analyzed using the model proposed by Lamb, which provides an accurate description of the kinetics of recovery of rod sensitivity in normal subjects after adapting lights that cause bleaches ranging from 1% to >99% of the rhodopsin originally present.13"15'22 The scheme postulates that the control of rod sensitivity results from the persistent presence of small amounts of R* (the active form of photolyzed rhodopsin) after extinction of the adapting light. Lamb proposed that the R* is produced from three relatively long-lived rhodopsin photoproducts S^ linked in a series of sequential firstorder reactions, each of which is weakly reversible:

Rhodopsin ----• R* = =

k21

s*.

Interconversion of S2 and S3 is rate limited, with a half-saturating value for S2 of S2sai, and rhodopsin is regenerated from S3 with a first-order rate constant k34. The photoproducts, which are not explicitly identified, could include one or more forms of phosphorylated opsin22 and/or opsin to which arrestin is bound.23'24 The model provides a quantitative description of the kinetic abnormalities observed in several forms of RP, 1415 with an accuracy that is unattainable using conventional schemes for rod dark adaptation.2'2(> It further enables various time domains (each associated with the relative abundance of one of the species, Sj) during dark adaptation to be identified. As a result, when the kinetics are abnormal, the extent to which each of these domains is affected can be individually assessed. Solutions of the first-order differential equations describing the model were obtained by numerical integration using the Runga-Kutta method,27 and, for each subject, values for the parameters were

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obtained by minimizing the errors of the fits to the rod recovery data for all three bleaches. Imaging fundus reflectometry was carried out using instrumentation and methods already described.28"30 Rhodopsin losses in the patients were determined by comparison of their double difference values (at 520 nm) with those from normal subjects at matched retinal locations. To study the relationship between the rhodopsin levels and rod-mediated sensitivity, dark-adapted static perimetric measurements with the 500-nm stimulus were made at up to 25 loci within the retinal region tested with fundus reflectometry.

RESULTS Figure 1 shows fundus photographs and results of visual- field and ERG testing in patients MDl and RP1. Patient MDl has macular dystrophy with yellow lesions in the central retina of each eye. Best-corrected visual acuities were 20/200 in the right eye and 20/30

in the left eye, and kinetic visual fields were normal except for a small relative central scotoma. With static perimetry, rod thresholds were abnormal at fixation and at a few other loci outside the central visual field; cone thresholds were only abnormal at fixation. The rod ERG b-wave was reduced in amplitude (59 ;/V) and delayed in timing (101 msec); amplitudes of both the a-wave (109 fiV) and the b-wave (211 fiV) of the mixed cone-rod ERG were reduced; cone ERG amplitudes were reduced (32 and 38 /iV for 1 Hz and 29 Hz, respectively), and timing was delayed to the flicker (37 msec). Patient MD2 also has macular dystrophy with yellow lesions in the central retina, visual acuity of 20/ 20 and 20/30 in the right and left eye, respectively, normal kinetic fields, a few abnormal rod thresholds but no abnormal cone thresholds with static perimetry, and normal rod, mixed cone-rod and cone ERGs. Patient RP1 has the ophthalmoscopic hallmarks of retinitis pigmentosa with pigmentary changes across most of the peripheral retina and relative sparing of the central retina. Visual acuity was 20/30 in each eye, and kinetic fields were limited to a central island of

Gly167Asp Gly167Asp

Normal

Pro210Ser

Gly167Asp

Pro210Ser

Kinetic perimetry

Pro210Ser

Rod

Eccentricity (deg)

FIGURE 1. Comparison of phenotypes of patients with Glyl67Asp and Pro210Ser RDS mutations by ophthalmoscopic appearance (a), electroretinography (b), and kinetic and static perimetry (c). (a) Fundus photographs of patient MDl showing macular lesion (left) and of patient RP1 showing pigmentary retinopathy outside the macula (right), (b) Rod, mixed cone and rod, and cone ERGs for 1 Hz. and flicker in a representative normal subject (left), patient MDl (center), and patient RP1 (right). Arrows indicate stimulus onset, (c) Psychophysical results from patients MDl (left) and RP1 (right): kinetic perimetry (top); static perimetry for cones (center), and rods (bottom). Static perimetry data are shown as grey scales of sensitivity loss. Scales have 16 shades representing sensitivity losses of 0 to 2.5 log units for cones and 0 to 5.4 log units for rods. Physiological blind spot is shown as a black square at 12° in the temporal field.

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function separated from a peripheral island by an absolute midperipheral scotoma. Static perimetry showed abnormal rod and cone function in the central and peripheral islands. Rod and cone ERGs were not detectable. Patient RP2 had no ophthalmoscopic abnormalities, 20/20 acuity in each eye, and normal kinetic and static perimetry results. The rod ERG b-wave was slightly reduced in amplitude (154 /iV) and delayed in timing (88 msec); the amplitudes of the a-wave (258 /xV) and b-wave (289 j*V) of the mixed cone-rod ERG fell within the normal limits; the cone ERGs were slightly reduced in amplitude (30 MV and 40 fiV for 1 Hz and 29 Hz, respectively) but were normal in timing. Dark adaptation data for patients MD1 and MD2, measured at 12° in the inferior field, are shown in Fig. 2a, together with those in three normal subjects. In normals, recovery of rod sensitivity to prebleach levels is complete after about 55 minutes. In both patients, the recovery of rod sensitivity followed a similar time course that was abnormally delayed, requiring about 90 minutes. Dark adaptation in patient MD1 at a more peripheral location (30° in the nasal field) after the same bleaching exposure also followed the same time course (Fig. 2a). Final sensitivities in patients MD1 and MD2 were within 0.3 log]() units of those of the normals at each of the test locations. Analysis of the rodmediated portions of the recovery of sensitivity in patients MD1 and MD2 using the scheme proposed by Lamb 1 ' 14 yielded similar parameters for both patients, so a single curve is shown to describe the data. A single fitting curve is also used to describe the normal data. Recovery of cone sensitivity in patients MD1 and MD2, measured at 30° in the nasal field, are shown inset in Figure 2a, together with data from two normals. Both patients' cones showed similar recovery kinetics and were slower than normal. Results of dark adaptometry from patients RP1 and RP2 are illustrated in Figure 2b, together with data from two normals. In patient RP2, the recovery of rod sensitivity (measured at 30° in the nasal field) was also abnormally slow, requiring about 90 minutes to return to its prebleach level (which was within 0.2 log units of normal). The solid curve in Fig. 2b is the same curve as was used to fit the data of patients MD1 and MD2 and illustrates the similarity of recovery in patients RP2, MD1, and MD2, independent of mutation. Because of patient RP1 's extensive loss of peripheral rod function, dark adaptometry was carried out at 6° inferior. The overall time required for dark adaptation in patient RP1 was similar to that of patient RP2, but the onset of the rod-mediated branch of the recovery, though less well denned, appeared to be more delayed, and final threshold was elevated from normal by >1.0 log units. Recovery of cone sensitivity in patient RP2 was slightly delayed from normal (Fig. 2b, inset), but that in patient RP1 was considerably slower.

PB0

20

40

60

Minutes FIGURE 2. Dark adaptation, measured at 500 nm, after a full bleaching exposure, (a) In patients with the Glyl67Asp mutation (unfilled symbols): patient MD1 (squares) and patient MD2 (triangles) tested at 12° below fixation, and patient MD1 (circles) tested at 30° in the nasal field. Filled symbols are from three normal subjects. Curves are results of kinetic analyses of the rod recovery data using the scheme proposed by Lamb,1H based on the parameters given in Table 1. (Inset) Recovery of cone sensitivity, measured at 650 nm, of patients MD1 and MD2 and two normals shown with expanded time scale, (b) In patients with the Pro210Ser mutation (unfilled symbols): Patient RP2 (circles) tested at 30° in the nasal field, and patient RP1 tested at 6° below fixation (triangles). Filled symbols are data from two normals tested at 30° in the nasal field. Broken dashed curve is the fit obtained for patient RP1 from kinetic analysis; solid and dashed curves are from (a) displaced vertically to optimize registration with the data. Recovery of cone sensitivity of patients RP1 and RP2 are shown (inset) on expanded time scale. PB = prebleach thresholds.

The parameters of the kinetic scheme13 giving the best fits to the patient rod dark-adaptation data in Figure 2, and also to adapting exposures that bleached part of the pigment in each subject (data not shown), are given in Table 1. Visual pigment levels were measured with imaging fundus reflectometry in patients MD1, MD2, and RP2. Measurement was over a circular area of retina


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TABLE l. Subject Glyl67Asp MD1

Parameters* Describing the Kinetics of Rod Dark Adaptation k32 k34 §s2sal tk2, .05 .04

.00082 .0003

.010 .0072

.00006 .000065

.0019 .0016

.11 .15

RP1 RP2

.04 .04

.00045 .0005

.0062 .0093

.00007 .00007

.0009 .0016

.125 .125

Vitamin A Day 0 Day 1 Normal

.04 .04 .045

.0006 .0006 .0007

.0045 .0085 .0125

.00004 .00005 .000055

.0015 .0018 .00275

.13 .13 .13

MD2 Pro210Ser

* Parameters obtained using scheme proposed by Lamb.1' In all cases, kl0 (see Methods) was treated as invariant from normal and the final threshold, relative to that in normals, was used for estimating the number of rhodopsin molecules present. f All kjj values are in units of s~'. | This parameter depends on data obtained immediately following weak bleaching adaptation and is subject to variability. § Sjjslll is the half saturating concentration of S2.

of diameter 15°. In patient MD1, the measurement area included part of the well-defined central macular lesion: outside that area, measured density changes were only mildly reduced from normal, whereas within it they were substantially lower (Fig. 3a). Figure 3b shows the spectral characteristics of the measured pigment from the two areas. Away from the lesion, the data match those normally observed for rhodopsin29 whereas within it the maximal double density change is shifted to longer wavelengths. Such a shift is consistent with an increased relative contribution by the medium- and long-wavelength cones to the measured density changes, but it may also result in part from the increased scatter in this more reflective region of the fundus.28'30 The reductions in measured density levels in patient MD1 in relation to his reduced rod sensitivity at the same points in the visual field are shown in Figure 3c. The data points lie close to the line illustrating the predicted relationship for rod sensitivity losses caused by decreased light absorption as rhodopsin levels diminish.2931 In patient MD2, the imaging fundus reflectometry measurement area (similar in location and size to that in patient MD1) included small yellow lesions. Outside these areas of abnormal lesions, double-density levels had spectral characteristics consistent with those of rhodopsin (Fig. 3b). Generally, measured levels were more than half the normal values and followed the same relationship to visual sensitivity deficits as in patient MD1 (Fig. 3c). There were, however, several small and clearly defined visual pigment dropouts in locations corresponding to the yellow lesions. Within these, pigment levels were only about 25% of normal, and the wavelength of maximum densities was shifted to longer wavelengths, as illustrated in the example shown in Figure 3b. In patient RP2, imaging fundus reflectometry was carried out on a region cen-

tered on 30° in the nasal field, where the dark-adapted rhodopsin level was found to be about 75% of normal;2931 its relation to patient RPl's slightly reduced rod sensitivity at that location was similar to that of patients MD1 and MD2 (Fig. 3c). In patients MD1 and MD2, recovery of rhodopsin double density after essentially complete pigment bleaching followed similar abnormal biphasic patterns (Fig. 3d), with a relatively rapid early phase then a slow increase to the final level. The first phase was slightly slower than the recovery in the control subject shown in Figure 3d, which, as in other normals, can be described by a single exponential with a time constant of 6 minutes.29'31

DISCUSSION Wide variations in clinical phenotypes can occur within and between mutations in the peripherin/RDS gene: Diseases associated primarily with loss of peripheral function and classified as Rp 7 8 1 1 1 2 O r a variant thereof,9 or diseases of the macula10"12 have been reported. Such disparities are illustrated by the patients described here. The fundus appearance, with narrowing of vessels, and pigmentary retinopathy observed in patient RP1, with the codon 210 mutation, are typical of RP and contrast strongly with the maculopathy observed with the codon 167 mutation.10 The basis of such radical differences is unknown. The predicted structure of the peripherin molecule in the photoreceptor disk membrane2 would place both mutations in the same luminal loop of the protein, suggesting that the domain of the molecule is not the basis for the different patterns of retinal degeneration.10 Potential explanations based on the supposition that there are interactions between peripherin and other protein(s) specific to rod or cone outer segments (and that might

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450

80

60

40

500

550 600 Wavelength (nm)

650

700

20

% Rhodopsin

Minutes

FIGURE 3. Fundus reflectometric data from patients MDl, MD2, and RP2. (a) Map of the circular measurement area in patient MDl showing dark-adapted levels of pigment densities; low levels close to fixation correspond to the clearly delineated central lesion in this subject, (b) Spectral dependence of the measured double densities in patients MDl and MD2 at 12° below fixation (open symbols), at a location corresponding to yellow pigment deposition in patient MD2 (filled circles) and within the lesion in patient MDl (filled triangles). Curves are rhodopsin spectra from normals, scaled for best fit. (c) Measured pigment levels, relative to normal, in relation to rod sensitivity loss at corresponding locations in patient MDl (triangles), patient MD2 (circles), and patient RP2 (square), (d) Recovery of density differences, measured at 520 nm, after full rhodopsin bleaches in patient MDl (triangles), patient MD2 (circles), and a normal subject (squares) at 12° below fixation. Curve is single exponential.

depend critically on the integrity of the polypeptide sequence within the lumen) have been proposed 3 " 12 but have no experimental foundation as yet. Despite showing different clinical phenotypes, the Glyl67Asp and Pro210Ser mutations lead to the same psychophysical abnormality of photoreceptor function, delays in rod and cone dark adaptation. The kinetics of the recovery of rod sensitivity in the mildly affected representatives of both mutations proved to be well described using the scheme proposed by Lamb13 (Fig. 2). The similarities between the data obtained from two mutations, and therefore of the parameters yielded by the analysis (Table 1), contrast with what is observed in patients with adRP caused by mutations in the rhodopsin gene..In the rhodopsin mutations that show abnormal rod dark adaptation, the kinetic abnormalities have been found to differ substantially between mutations, 1415>21'32>33 as would be expected if the effects on rod function were specifically determined by the site of the mutation on the

rhodopsin molecule in each of them. In the patients with Glyl67Asp and Pro210Ser peripherin/RDS mutations, the two parameters which are notably and consistently abnormal are k23 and k34 (Table 1). The value of k2;, can be estimated with good precision because it is determined by the slope of the recovery in the period immediately after the rod-cone break. In each case, it is significantly lower than in normals13'143'1 by an amount that leads to rod sensitivities more than 1.0 log units abnormal in the patients after about 20 minutes of dark adaptation (Fig. 2). Similarly, the reduced k34 values of the patients reflect their extended final phase of rod recovery; this reaction is most likely to be associated with the final stage of the visual cycle, the regeneration of activatable rhodopsin.1322 In patients MDl and MD2, in whom it was possible to obtain rhodopsin regeneration data, delayed kinetics were indeed found (Fig. 3d), supporting the idea that the psychophysical abnormalities are linked to perturbations in the rod visual cycle. In patient RP1, whose

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loss of rod function was most advanced, the parameters required to produce a fit were similar in type to those of patients MD1, MD2, and RP1, but k34 showed greater abnormality, suggesting that as the disease process progresses, the degree of dysfunction becomes more extreme. The specific adaptational changes revealed by the kinetic analysis and the possible involvement of the visual cycle suggested by the slower-than-normal kinetics of rhodopsin regeneration are strongly reminiscent of the anomalies observed in mild systemic vitamin A deficiency in humans.1617 Intriguingly, abnormalities of adaptation that are apparently similar to those described in this study have also been reported in patients with age-related maculopathy.35 There are quantitative differences, however, from other diseases that involve abnormal recovery of rod sensitivity, such as adRP with rhodopsin gene mutations,2 '-32-33 fundus albipunctatus,3637 Stargardt's disease,38 and Oguchi's disease.36-37 Dark adaptation data obtained from a subject with chronic liver disease who suffered from visual symptoms resulting from vitamin A deficiency, obtained both immediately before (day 0) and 16 hours after (day 1) oral supplementation with vitamin A, are shown in Figure 4. Before supplementation, the time course of dark adaptation was similar to those seen in vitamin A-deficient patients in previous studies1617 and also to that in patient RP1. The rod-cone break occurred at about 30 minutes, substantially later than normal, and complete recovery of rod sensitiviy, which

0

cone adaptation

1

?

2

;|

3

c 0) C/5

4

PB 0

20

40

60

80

Minutes FIGURE 4. Dark-adaptometry data obtained at 30° in the nasal field from a subject with systemic vitamin A deficiency before (triangles) and 16 hours after (circles) oral supplementation (with 15,000 IU as palmitate); broken curves are fits obtained from kinetic analysis,13 solid curve isfitfor normals, from Figure 2. (Inset) Recovery of cone sensitivity of same subject and two normals (filled symbols) shown with expanded time scale.

was within 0.3 log units of normal, took about 80 minutes. Recovery of cone sensitivity was also abnormally prolonged and had a time course similar to that of patient RP1. After supplementation, rod adaptation was substantially improved and followed a time course close to that in patients MD1, MD2, and RP2. The kinetics of cone recovery were also faster and were similar to those in patients MD1 and MD2. These similarities were reinforced by kinetic analysis of the rod dark-adaptation data of the vitamin A-deficient subject. The major anomalies observed (Tabe 1) were in parameters k23 and k34, as in the patients with peripherin/RDS mutation, and the degree of abnormality was reduced after supplementation. The similarities in the abnormalities in receptor adaptation, both between the mutations and to those in vitamin A deficiency, raise the possibility that peripherin, in addition to its structural role(s),2'4 plays some part in maintaining the normal functioning of the visual cycle. This possibility gains support from another characteristic that appears to be commonly observed in disease associated with peripherin/RDS mutations—white9 or yellowish deposits1011 at the level of the pigment epithelium that are also observed in systemic vitamin A deficiency.39 Degenerative changes observed in animal studies of chronic vitamin A deficiency are also consistent with some of the present observations on patients with peripherin/RDS gene mutations. These include outer segment shortening and disorganization in both rods and cones,40"42 which would lead to the reduced rhodopsin levels measured in the mildly affected patients (Fig. 4), and, as disease progresses, final dark-adapted sensitivities substantially below normal, as seen in patient RP1. Irreversible cone loss in the central retina has also been observed.4'4143 Details of any mechanism linking slowed adaptation to a defect in the visual cycle caused by mutation in the peripherin/RDS gene will necessitate further studies of normal function, as well as animal models of the human diseases,4"6 to establish whether the link is direct or results from degenerative changes in the photoreceptor. The specific phases of abnormal dark adaptation suggest that one or more steps involving the visual cycle is modified. Candidates include the transport-inclusion mechanism of retinoids into photoreceptor membrane for rhodopsin regeneration,44 the later stages of rhodopsin photolysis (such as its phosphorylation23 and interaction with arrestin),24 or the activity of the retinal reductase.24'44 Possible functional roles for peripherin in reactions such as these merit further investigation. The effects of different amino acid substitutions within the peripherin protein in such functional studies could also throw light on other areas, for instance, links between dysfunction and degenerative processes.

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Peripherin/RDS Mutations and Retinal Function Key Words peripherin/RDS, macular degeneration, retinitis pigmentosa, rod adaptation, rod photoreceptors

13.

Acknowledgments

14.

The authors thank Mrs. D. Slaughter, Ms. K. Stewart, and Mrs. B. Koernig for coordinating this study; Dr. X. Sun and Mr. D. Azevedo for help with data acquisition; and Mr. B. Eisner for assistance with data analysis.

15.

References 1. Molday RS, Hicks D, Molday L. Peripherin: A rimspecific membrane protein of rod outer segment discs. Invest Ophthalmol Vis Sci. 1987; 28:50-61. 2. Connell G, Molday RS. Molecular cloning, primary structure and orientation of the vertebrate photoreceptor cell protein peripherin in the rod disc membrane. Biochemistry. 1990; 29:4691-4698. 3. Arikawa K, Molday LL, Molday RS, Williams DS. Localization of peripherin/rds in the disk membranes of cone and rod photoreceptors: Relationship to disc membrane morphogenesis and retinal degeneration. J CellBiol. 1992; 116:659-667. 4. Travis G, Sutcliffe JG, Bok D. The retinal degeneration slow (rds) gene product is a photoreceptor disc membrane associated glycoprotein. Neuron. 1991; 6: 61-70. 5. Travis G, Brennan MB, Danielson PE, Kozak CA, Sutcliffe JG. Identification of a photoreceptor-specific mRNA encoded for the gene responsible for retinal degeneration slow (rds). Nature. 1989;338:70-73. 6. Connell GJ, Bascom R, Molday L, Reid D, Mclnnes RR, Molday RS. Photoreceptor cell peripherin is the normal product of the gene responsible for the retinal degeneration in the rds mouse. Proc Natl Acad Sci USA. 1991;88:723-726. 7. Farrar JG, Kenna P, Jordan SA, et al. A three-basepair deletion in the peripherin-RDS gene in one form of retinitis pigmentosa. Nature. 1991;354:478-480. 8. Kajiwara K, Hahn LB, Mukai S, Travis GH, Berson EL, Dryja TP. Mutations in the human retinal degeneration slow gene in autosomal dominant retinitis pigmentosa. Nature. 1991; 354:480-483. 9. Kajiwara K, Sandberg MA, Berson EL, Dryja TP. A null mutation in the human peripherin/RDS gene in a family with autosomal dominant retinitis punctata albescens. Nature Genet. 1993; 3:208-212. 10. Nichols BE, Sheffield VC, Vandenburgh K, Drack AV, Kimura AE, Stone EM. Butterfly-shaped pigment dystrophy of the fovea caused by a point mutation in codon 167 of the RDS gene. Nature Genet. 1993;3:202207. 11. Wells J, Wroblewski J, Keen J, et al. Mutations in the human retinal degeneration slow (RDS) gene can cause either retinitis pigmentosa or macular dystrophy. Nature Genet. 1993;3:213-218. 12. Weleber RG, Carr RE, Murphey WH, Sheffield VC, Stone EM. Phenotypic variation including retinitis pigmentosa, pattern dystrophy, and fundus flavimacu-

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