Autosomal-dominant Retinitis Pigrnentosa Associated with an Arg-135-Trp Point Mutation of the Rhodopsin Gene

Autosomal . . dominant Retinitis Pigmentosa Associated with an Arg . . 135 . . Trp Point Mutation of the Rhodopsin Gene Clinical Features and Longitudinal Observations Mario R. Pannarale, MD, 1 Barbara Grammatico, BS, 2 Alessandro Iannaccone, MD, 1 Renato Forte, MD, 1 Carmelilia De Bernardo, BS, 2 Luisa Flagiello, BS / Enzo M. Vingolo, MD, PhD,l Giuseppe Del Porto, MD2 Purpose: To report the clinical and functional characteristics of patients affected with autosomal-dominant transmitted retinitis pigmentosa (adRP) from a large Italian pedigree in which a point mutation predicting the Arg-135-Trp change of rhodopsin was identified by polymerase chain reaction-single-strand conformation polymorphism analysis. Methods: Seven patients, ranging in age from 6 to 41 years, underwent a full clinical ophthalmologic evaluation, kinetic visual field testing, and electroretinographic testing. Results: In agreement with previous reports, this rhodopsin mutation yielded a particularly severe phenotype, both clinically and functionally. The evaluation of patients from this pedigree in the first and second decade of life demonstrated that retinal function is still electroretinographically measurable at least until 18 years of age, although reduced to 2% to 4% of normal. Longitudinal measures showed that the rate of progression of the disease was unusually high, with an average 50% loss per year of electroretinographic amplitude and visual field area with respect to baseline. Later in the course of the disease, macular function is also severely compromised, leaving only residual central vision by the fourth decade of life. Conclusions: The phenotype associated with mutations in codon 135 of the rhodopsin molecule appears to have an unusually high progression rate and yields an extremely poor prognosis. These distinctive features make the Arg-135-Trp phenotype substantially different from the general RP population, and also from many of the other adRP pedigrees with known rhodopsin mutations reported to date. Ophthalmology 1996; 103:1443-1452

Originally received: August 8, 1995. Revision accepted May 16, 1996. I Center for Inherited Degenerative retinal Disorders, University "La Sapienza," Rome, Italy. Medical Genetic Section of the Experimental Medicine Department, University "La Sapienza," Rome, Italy.

2

3

International Institute of Genetics and Biophysics (11GB), Naples, Italy.

Dr. Iannaccone currently is on a research fellowship at the l)niversity of Pennsylvania, Scheie Eye Institute, Center for Hereditary Retinal Degenerations, Philadelphia. Performed in partial fulfillment of the requirements of Dr. A. Iannaccone for a research doctorate at the University "La Sapienza." Reprint requests to Prof. Giuseppe Del Porto, MD, Medical Genetic Section

Several different rhodopsin gene mutations have been identified in the last years in pedigrees with autosomaldominant retinitis pigmentosa (adRP). Defining the associated phenotypes has become increasingly important to identify the clinical counterpart to the different functional abnormalities of the photopigment molecule.'-27 This is particularly true if one considers the notable clinical and functional heterogeneity associated with the different genotypes (Table 1). In vitro expression studies on different mutant rhodopof the Experimental Medicine Department University "La Sapienza," c/o Ospedale L. Spallanzani, Via Portuense 292 - 00149 Rome, Italy.

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~ ~ ~

lmradiscalt Intradiscalt Intradiscal Intradiscal lntrad isca l Intradiscal Intradiscal Imrad iscal Intradiscal T ransmemhrane ( intradiscal)t Transmembrane (cytoplasmic}t T ransmembrane (cytoplasmic):j: Transmembrane (cytoplasmic):j: Transmembrane (cytoplasmic):j: T ransmembrane T ransmembrane (ll-cis-ret)§ Cytoplasm (I loop) Cytoplasm (IV loop) Cytoplasm (IV ol op) Cytoplasm (C-terminus ) Cytoplasm (C-terminus) Cytoplasm (C-terminus) Cytoplasm (C-terminus) Cytoplasm (C-terminus) Cytoplasm (C- terminus ) Cytoplasm (C-tenninus)

Alteration Site Preserved Well preserved Well preserved Well preserved Moderately impa ired Moderately impaired Well preserved Well preserved Well preserved Moderately impaired

Visual Acuity

Fairly preserved

Impaired

Fairly p reserved

Fairly preserved

Fairly preserved

Fairly preserved

Fairly preserved

Fairly preserved

Earl y (I decade) Impaired

Diffuse to all quadrants

Diffuse to all quadrants

Impa ired

Early « 11 yrs)

Diffuse to all quadrants

Mostly diffuse t o lal quadrants

Diffuse to all quadrants

Diffuse to all quadrants

Variab le (none to diffuse)

Variable (none to diffuse)

Variable (none to diffuse)

Diffuse to all quadranrs

Fairly p reserved

)

Sectoral (inferior and nasal) Diffuse to all quadrants

Sparse but diffuse (360 0

Diffuse to all quadrants

Diffuse to all quadrants

Sparse and sectoral (inferior)

Sectoral (infer ior) Sparse and sectoral (inferior) Sectoral (inferior and nasal) Sectoral ( inferior) Sectoral (inferior) Sparse but diffuse (360°) Sparse but diffuse (360°) Sectoral ( inferior) Sectoral (inferior) to diffuse (360°) Diffuse to all quadrants

Pigmentary Changes

Early (I-II decade) Variably impaired

Early (I decade)

Early (I-II decade) Fairly preserved

Early (II decade)

Early (ll decade )

Late

Late

Early (I-ll decade) Well preserved Early ( birth ) Impaired

Early (childhood )

Early

Early

Early (I-ll decade) Well preserved

Varia hie Late (> ll decade) Widely v ariable Late (IV decade) Early (I- II decade) Early (I decade ) Variahle (6- 30 yrs) Late (> II decade) Early (I decade) Early (I decade)

Age of Onset

* Point mutations, stop codon mutations, deletions, etc. t G yl cosylated residues in the in tradiscal domain. :j: Side of the disc membrane toward wh ich transmembrane mutations are located. § Mutation at the attachment site for ll-cis-retinal.

Pro-34 7_Ser

ls

Pro-3 47-Arg 14

Pro-34 7

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Val-34S- Leu26

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Arg-13 5_Leu 6•9

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Asn- 15-Ser" '" T hr- 17_Met6." .'9 Pro_ 23_His l ,5.8 G ly-106-Arg 12 G ly- 182-Ser" Cys- 187-TyrZ7 G ly- 188-Arg '6 Asp- 190-Asn 13 Asp-190-T yr13 Leu-40-Arg'O

Rhodopsin Alterations*

Benign Benign Fairly henign Benign Benign Severe Fairly benign Ben ign (Severe ?) Severe

Prognosis

Severe reduction

Severe reduction

Moderate to severe reduction

Moderate to severe reduction

Mild to severe reduction

Normal to severe reduction

Mild to severe reduction

Mild to severe sensitivity loss

Mild reduction

Mild to severe reduction

Mild reduction (ring scotomas) Severe reduction

Benign Severe

Moderately severe

Fairly benign

Severe ly reduced

Severely reduced

Severely reduced

Severely reduced

Severely reduced

Variab ly reduced

Severe

Severe

Severe

Q uite severe

Quite severe

Moderately severe

Moderately reduced Moderately severe

Severely reduced

Mildly reduced

Moderately reduced Fairly benign

Mildly reduced Severely reduced

Moderately reduced Fairly benign

Moderate to severe reduction

Severe

Severe Severely reduced

Severely reduced

Moderately reduced Benign

M ild ly reduced Mildly reduced Moderately reduced Moderately reduced Variably reduced Severely reduced Moderately reduced Mildly reduced Severely reduced Severely reduced

ERG Amplitude

Severe reduction

Severe reduction

Mild re duction ( > superior)

Mild reduction (Superior defect) Mild reduction (Superior defect) Mild reduction (superior defect) Mild red uction (superior defect) Mild reduction (superior defect ) Severe reduction Moderate to severe reduct ion Mild reduction (superior defec t) S uperior defect to severe reduction Severe reduction

Visual Field Areas (V4e)

T able 1. Summary of the Phenotypes Associated with Different Rhodopsin G ene Alterations

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Pannarale et al . Arg-135- T rp Rhodopsin Mutation in adRP Figure 1. Pedigree 88/46 with autosomal-dominant transmitted retinitis pigmentosa in which a point mutation (Arg-135-Trp) of the rhodopsin gene eventually was identified by single-strand conformation polymorphism analysis. Filled symbols = affected individuals carrying the mutation; / = proband; slashed symbols = deceased. Indicated are also the results of linkage analysis with the RHO (MFD1) marker, which pointed to the presence of a rhodopsin gene mutation (0, informative allele) , initially not detected by polymerase chain reaction hydrolink gel analysis (see Results section for further details).

II III IV

V 2

3

4

5

sins have allowed a better classification of rhodopsin gene defects on biochemical grounds, identifying two major classes of mutations 28 : class I, resembling the wild-type opsin in their accumulation to high levels in the plasma membrane and their ability to join in vitro to II-cis retinal to form a photo labile visual pigment: these mutations tend to cluster near the carboxyl terminus. Phenotypes associated with this class of mutations tend to be consistently severe. Class II mutations accumulate to much lower levels, are inefficiently transported from the endoplasmic reticulum to the plasma membrane, and produce little or no photolabile pigment upon incubation in vitro with II-cis retinal: this class of rhodopsin mutants is likely to be defective in protein folding and/or stability, and has been associated with various clinical pictures, ranging from mild (sector) to severe forms ofRP. 23 .28 This last class of rhodopsin mutants can be subdivided further in class IIa, which shows prevailing intracellular accumulation, and class lIb, which also shows significant cell surface localization. 28 Class II rhodopsin mutants cause different degrees of RP severity. Mutations affecting the intradiscal domain seem to yield the better prognosis, whereas transmembrane mutations are associated with more variable clinical pictures, apparently depending on the biologic relevance of the involved residue,z9 Here we describe the phenotype of a large Italian adRP pedigree in which a class lIb point mutation of the rhodopsin gene at codon 135 (Arg-135Trp) has been demonstrated. The precise location of this residue is not completely known. In this study, we considered it in the transmembrane domain at the edge of the cytoplasmic side, as reported by Jacobson et a1. 6 Data are compared with findings from earlier reports describing the phenotypes yielded by mutations at the same codon6 ,9 and with those of another Italian pedigree with a class lIb mutation (Gly-I88-Arg) that we have previously described. 16 Longitudinal measures on some of the younger patients with the Arg-135-Trp mutation were also possible, allowing estimation of the progression rate of RP in patients carrying this defect. We also updated a summary of most of the available data on phenotype-genotype

I

12

13

14

15

16

correlations derived from a previous publication 16 to get an overall view of the current knowledge in this field.

Patients and Methods Patients The five-generation pedigree studied here was Sicilian in origin, and was ascertained from a proband with RP (lV3). Twenty-three affected individuals have been identified on historical bases. Of these, seven (IV-3, IV-5, V-7, V8, V-lO, V-13, and V-14), ranging from 6 to 41 years of age, have been investigated according to previously published criteria,16 which are summarized in the foll owing paragraph. The pedigree shows an autosomal-dominant pattern of transmission (Fig 1).

Clinical and Functional Ophthalmologic Procedures A detailed medical history was taken to define the age of onset of symptoms of the disease, with particular interest in night vision impairment, reduced side vision, light aversion, and progressive loss of visual acuity. Best-corrected far and near visual acuities were assessed with a standard Snellen chart and astigmometric charts modifi ed according to Pannarale.30 Anterior and posterior segment conditions were classified according to criteria listed in Table 2. Lens opacities (posterior subcapsular cataracts) were biomicroscopically graded according to previously published criteria31 modified from Fishman et al. 32 Vitreal aspects were biomicroscopically graded according to the classification shown in Table 2, as previously reported. 16 Fundus examination was performed by indirect binoc ular ophthalmoscopy and slit-lamp biomicroscopy to classify retinal changes. The occurrence of macular alterations , such as epiretinal membranes, cystoid macular edema, macular atrophy, pigmentary changes, or subretinal neovascular membranes, also was recorded. Visual fields were tested by Goldmann kinetic perime-

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Volume 103, Number 9, September 1996

*E E Figure 2. Representative 0.5-Hz maximal electroretinographic responses from four subjects of this pedigree (right eye). Despite severe reduction in amplitude and delay of both a- and b-waves, responses still can be recorded (offline averaging of 80-100 iterations). Horizontal calibration = 25 mseconds. Vertical calibration = 2.5 ,.tV.

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try with 14e and V4e targets as previously reported. 16,33 The areas of the above targets were measured on conventional perimetry charts with a light pen and expressed in square centimeters, according to a previously described technique. 33 Scotomata within each isopter were measured and subtracted from the total area. Measures were compared with normal values and defined as percent of lower normal limits. Electroretinographic (ERG) analyses all were performed In dark-adapted conditions and included rod, mixed, and cone ERGs, All signals were recorded with dilated pupils and after 30 minutes of dark adaptation, After topical corneal anesthesia, Henkes-type corneal contact lens electrodes were placed on both eyes under dim red light and connected to a mechanical membrane suction pump to keep the electrodes stable on the corneal surface. 34 Low-noise recording techniques were used in patients with amplitudes less than 10 /LV, as described in detail elsewhere. 34 Electroretinograms were elicited with a lO-/Lsecond, 2-cd second/m2, white-flash Ganzfeld stimulation, For rod ERGs, a O.S-Hz repetition rate was used, attenuating the flash by 2.7 log units with neutral-density filters (Kodak Wratten, Eastman Kodak Co, Rochester, NY). Averaging of 80 to 100 iterations was performed. Maximal ERG responses (mixed) subsequently were recorded as above, with no attenuation of the stimulus. Finally, cone ERGs were recorded with a 30-Hz, whiteflash Ganzfeld stimulation (same intensity as above), av-

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351M

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0 0 0 0 4 4 4 4 0 0 0 0 0 0

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= posterior subcapsular cataract; 10L = intraocular l ens

OS

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00

OS

0.1 HM 0.4 0.6 1.0 0.7 0.9 0.6 0.7 O.S 0.7 0.8 0.5 0.5 1.0 0.9 1.0 0.7

Visual Acuity

= not

360 360 360 360 360 360 360 360 360 360 360 360 360 360 360 360 360 360 1 1 I I I 0 0 0 0 0 0

1 I I 1 0 0 1

:j: Patients V-7 n a d V-8 examined completely twice, in 1992 and 1993.

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2.8 NA 4.8 3.3 26.7 25.2 13.0 10.0 71.2 98.8 56.5 49.5 NA NA 31.8 26.7 11.0 8.5

V4 NR NR NR NR NA NA NA NA NA NA NA NA NA NA 4 4 NR NA

Rod b Ampl NR NR NR NR 12.75 13.75 6.50 5.50 NA NA 10.00 11 .50 NA NA 14.00 15.00 1 2.50 NA

bAmpl

43 51 60 59.5 NA NA 57 .5 58.5 NA NA 58 56 46 NA

b Peak

NA NA NR 1.08 NA NA 0.50 0.62 0.37 NA

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3 0-Hz

± 2 mseconds; b-wave peak

16 23 24 25 NA NA 23 20 NA NA 26 29 29 NA

a Peak

ERGt Maximal R esponse

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available.

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motions; NA

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= h and

Extension (0)

recordable; HM

Morphology

Visual Field* (area, %)

* Visual field s a re expressed as percent of the lower normality limit. Visual fi eld: peripheral limits, lower normality range: 620 cm t Rod ERG = amplitude (b ampl), lower normal li mit, 200 j1V; maximal 0.5-Hz ERG response, amplitude (b ampl) ; lower normal limit, 400 time: 45 ± 3 mseconds; cone 30-Hz ERG = ampli tude (amp!); lower normal limit, 50 j1V .

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Age (yrs )/Sex

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Ophthalmoscopic Findings

T able 3 . Summary of the C linical and Functional Findings from Affected Subj ects of th e 88/46 Pedigree (A rg-135-Trp rhodopsin mutation )

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Ophthalmology

Volume 103, Number 9, September 1996

eraging 60 to 100 samples. Offline analyses allowed separate averaging of groups of samples to verify the reproducibility of the signal. 34 Amplitude and peak time measurements were performed according to the international standards for clinical ERG. 35 Rod and mixed signals were defined as recordable when a reproducible waveform exceeding twice the value of the background noise was detected. Noise measurements were performed at each recording session as described elsewhere. 34 Cone 30-Hz signals were defined as detectable when a clear peak at 30 Hz could be observed by fast Fourier transform analysis, mutuating a published approach used for steady-state flash, visual-evoked potentials. 36 To obtain a sense of the rate of progression of retinal degeneration in this phenotype, best-corrected visual acuity (best eye) was plotted as a function of disease duration. This was calculated by subtracting the age of onset of symptoms from the age of the patients at the time of last examination. In addition, longitudinal comparisons were performed on visual fields and on ERG responses, when available. Therefore, yearly percentile losses of both residual visual field area and ERG amplitUde also were estimated.

Genetic Analyses Genomic DNA was extracted from peripheral blood lymphocytes using phenol-chloroform extraction. Polymerase chain reaction (PCR) was performed to amplify the five exons of the rhodopsin gene. The program used for PCR consisted of 30 cycles, including two successive steps at 94°C for I minute and at 63 °c for 2.5 minutes, respectively. The PCR products generated were run on ethidium bromide-stained hydrolink gel. 37 Linkage analysis was the second step of our molecular study. Nine affected and seven unaffected members of this pedigree were typed for microsatellite polymorphisms. The detection of the

mutation in the rhodopsin gene was achieved by PCRsingle-strand conformation polymorphism analysis. The rhodopsin gene was amplified by multiplex PCR. Exons 2, 3, 5, and the 5' -end of exon 1 were amplified as single segments; exon 4 and the 3' -end of exon 1 were first amplified as single segments and then digested with Bst Nl. Amplified DNA was heat-denatured, and single strand fragments were separated through nondenaturing 6% polyacrylamide gels, one with and one without 10% glycerol. After gel electrophoresis at room temperature, x-ray films were exposed to the dried radioactive gels according to Dryja et a1. 38

Results DNA Analysis Hydrolink gel analysis of PCR products failed to detect mutations in the rhodopsin gene. Therefore, linkage analyses were performed to define the possible involvement of other loci. However, linkage to the MFD2 microsatellite marker on chromosome 3 was demonstrated, suggesting that this pedigree actually was harboring a rhodopsin mutation. By PCR-SSCP analysis, a shift in exon 2 was found only in affected patients, whereas this was not observed in healthy and unrelated subjects. Exon 2 was amplified and cloned into a TA cloning vector (TA Cloning Kit, Invitrogen, San Diego, CA); positive clones were sequenced with an automatic sequencer. This resulted in the detection of a transition C-->T in codon 135, producing an Arg-->Trp single amino acid substitution. According to Sung et al' S28 criteria, this could be classified as a class lIb rhodopsin gene mutation. In addition, in some of the affected individuals a polymorphism of exon 3 of the RDS/peripherin gene also was observed.

Description of Patients 1.2 , - - - - - - - - - - - - - - - - - - - - - , R=0.939 p=O.0054

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Disease duration (years)

Figure 3. Best-corrected visual acuity plotted as a function of disease duration. O nly the eye with the best visual acuity is taken into account. The two parameters are closely correlated. Due to the early"tmset of the disease (3 - 6 years o fage), loss offoveal function is already very advanced by 40 years of age in patients carrying this rhodopsin mutation.

1448

Seven affected subjects-the proband (IV-3) and six additional family members (IV-5, V-7, V-8, V-lO, V-13 , and V-14)-between 6 and 41 years of age were examined. Detailed clinical and functional data for each patient are summarized in Table 3. The elder patients, with a more advanced stage of the disease (IV-3 and IV-5), had typical widespread retinal changes, affecting also the macular area. Visual acuity was reduced proportionally to the extent of foveal atrophy. Visual fields were limited to 10° to 15° around the fixation point with the V4e stimulus (2.8%-4.8% of lower normal limits). All ERG responses were undistinguishable from noise. Patients in their teens (V-7, V-8, and V-13) had a much better preserved visual acuity, but retinal changes were already remarkably advanced. Visual fields were variably reduced: with the I4e target, none of the fields exceeded 12° around the fixation point, whereas with the V4e target fields ranged from moderately narrowed (case V-8) reduced to the central 25° with small temporal islands (patient V-7 ; 25.5% on average). Signal averaging in all three patients allowed detection of maximal ERG responses that were 3% to 4% of normal amplitude and delayed

Pannarale et al . Arg-135-Trp Rhodopsin Mutation in adRP (Fig 2). Case V-13 also had a grossly delayed but still detectable rod response of 4 p,V (2% of the lower normal limit of 200 p,V). Cone 30-Hz responses were recordable in most instances, but not greater than 2.1 p,V (i.e., 4.2% of lower normal limits). The two subjects in their first decade of life (V-lO and V-14) also showed already clearly detectable fundus changes in all quadrants, with midperipheral retinal confluent flecks and grade 1 diffuse pigmentation. Retinal vessels also were already severely attenuated by this age. Visual acuities were moderately impaired, but this was mainly due to amblyopia (yet-uncorrected high hyperopia and astigmatism for case V-I 0, and anisometropia for case V-14). Visual fields yielded questionable results. Maximal ERG responses of approximately 3% of lower normal limits in amplitude were recordable from case V14 (Fig 2). Cone 30-Hz responses were detectable only by fast Fourier transform analysis, whereas rod responses were not distinguishable from noise. As shown in Table 3, some longitudinal observations were possible on cases V -7 and V-8. Over a I-year period, case V -7 showed an increased retinal pigmentation with macular atrophic changes that determined a mild loss in visual acuity. By this time, visual fields were reduced to less than 5° with I4e target « 1 %) and to 20° with V4e target, with no residual temporal island (11.5%, on average). No detectable change in visual acuity or in fundus appearance was noted for case V -8, but visual field loss increased substantially over a I-year period also in this patient (on average, visual field reduced to 53% of lower normal limits). There was a mean yearly visual field area loss of 46% for the V4e target and of 53% for the I4e target compared with baseline in these two patients. A similar rate of progression was seen for the maximal and cone ERGs of case V-7-on average, there was a 54% loss for the 0.5-Hz response and a 50% loss for the 30Hz response, respectively. In fact, the maximal ERG was reduced to approximately 1.5% of normal, and the flicker response was reduced to 2% to 2.5% of normal. A deterioration of the function of the remaining retina both receptoral and post-receptoral responses also was seen, with a greater delay of both a- and b-waves. Figure 3 shows visual acuity plotted as a function of disease duration. Data from case V-I 0 were not included due to bilateral amblyopia currently receiving treatment. All other patients showed impaired visual acuity proportionally to disease duration, with a high (r = 0.939) and significant (P = 0.0054) correlation of the two parameters. In view of the consistently early onset of the disease for all patients, and assuming this rate of visual acuity loss was similar for all of them, this would predict visual acuities in the 20/100 to 20/200 range by the fourth decade of life. Macular edema was never a cause of the visual acuity losses observed in this pedigree, whereas atrophic changes were seen as early as 16 years of age.

Discussion The evaluation of seven patients from a family with adRP, caused by a point mutation of the rhodopsin gene (Arg-

135-Trp), has allowed the accompanying clinical pattern to be defined. Patients described here showed no intrafamilial variability, and findings were consistent with the previously described features associated with mutations at the same codon in pedigrees from different geographic areas (i.e., United States 6 and Scandinavia9). Age of onset ranged from 3 and 6 years (Table 3). Night blindness and visual field narrowing were the first symptoms of all affected members. Results of fundus examination showed severely attenuated vessels and typical intraretinal bone spicule-like pigmentation affecting all four quadrants in every instance. Retinal changes were detectable as early as 6 years of age. Kinetic visual fields showed an early severe constriction nearly in all subjects to both the I4e and V4e targets. Rod responses were undetectable in all measurements, except in case V-13 (10 years of age), not exceeding 2% of the lower normal limit. These findings are consistent with perimetry and fundus reflectometry data from Jacobson et al,6 who showed no detectable rod function nor measurable rhodopsin in their patients with Arg-135 mutations. However, different from both previous studies that reported ERG responses invariably undistinguishable from noise,6.9 the evaluation of young subjects demonstrates that ERGs still can be measured in patients harboring the Arg-135-Trp mutation, at least until the second decade of life, provided that enough averaging and low-noise techniques are used. Maximal and 30-Hz white-flash stimulation yielded responses that, when measurable, were never greater than 3% to 4% of the lower normal limits. Although mutations at codons 135 and 188 belong to the same class of mutants (lIb), 28 the phenotype associated with the Gly-188-Arg mutation that we previously reported 16 was characterized by a diffuse retinal degeneration of much lesser severity. In fact, patients with this latter mutation showed severely narrowed visual fields only later in the course of the disease and had still recordable maximal ERG responses even in their 60s (2% of normal at 64 years of age). The youngest member of the examined patients with the Gly-188-Arg mutation (8 years old) showed a virtually normal fundus appearance and a 50% reduction of maximal ERG b-wave amplitude. This suggests that the distinction in different classes of mutants, taken alone, does not fully account for the observed clinical effect, likely depending on the biologic relevance of the involved residue to the function and three-dimensional array of the rhodopsin molecule. 39 In fact, a point mutation at codon 188 introduces a change near the cysteine residue at position 187 necessary to form and stabilize rhodopsin structure by establishing a disulfide bond. This is likely to disturb the formation of a functionally active rhodopsin by precluding the correct folding of the molecule. 16 ,27 This could explain both the relatively greater severity of the Cys-187 -Tyr27 phenotype compared with that of the Gly-188-Arg mutation,16 and why both of these are more severe than other class lIb mutations affecting less crucial residues (e.g., Thr-58Arg 4 ,7 or Gly-106-Arg ' 2 ). Mutations at position 135 have the potential to determine a greater clinical severity. In fact, the glutamate at position 134 is an important interaction site with transducin, because it provides a proton

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acceptor group to allow light- or ligand-dependent receptor activation. 40 The peculiar severity due to mutations at codon 135, already suggested in previous investigations,6,9 was verified also in the current study. Table 1 summarizes the clinical and functional features of the data on genotype-phenotype correlations for 26 rhodopsin mutations. 1-27 We have subdivided case reports according to the involved rhodopsin molecule domain (i.e., intradiscal, trans-membrane, and cytoplasmic). Based on some of the reported key parameters (age of onset, visual acuity, retinal pigmentary changes, visual field and ERG impairment, and progression over time), we also proposed an overall prognostic judgment for the phenotypes associated with each genotype. Data demonstrate that adRP is clinically heterogeneous. The severity of the disease varies in a complex manner, depending not only on the biologic relevance of the involved residue, but also on the rhodopsin domain affected, the latter concept being already proposed by other investigators. 28 ,41 ,42 Nearly all mutations found in the intradiscal domain show a benign prognosis, 1.5,6,8,11 ,13,16.19,21,23 and several also have a sectoral pattern of dysfunction. The main exception reported to date is the Cys-187 -Tyr - related phenotype?7 Mutations at the level of the cytoplasmic domain show a more consistently severe phenotype with diffuse retinal clinical and functional changes, with an onset within the second decade of age. 2,3,6, IO,14,J5,26 Different from this general behavior are the phenotypes determined by deletions,18,22 truncations,6,25 and alterations at splice sites. 20,25 These discrepancies have not been fully explained yet, and are likely to be related to the complex interactions of the carboxyl-terminal region during the phototransduction cascade. The sparing of the highly conserved valine and proline residues in positions 345 and 347, respectively, could possibly explain the milder phenotype shown by patients with the 341 to 343 deletion. ISIt also has been recently experimentally demonstrated that a truncation distal to Cys-316 does not affect transducin activation,43 and that an intact carboxyl terminus is essential for phosphorylated rhodopsin to interact with arrestin.44 Finally, mutations at the transmembrane level show a wide clinical and functional variability (both sectoral and diffuse patterns), ranging from benign to severe forms. 4,6.7,9,J3,20,24 As discussed above, this appears to correlate with the specific role of the involved residues, We also were able to make longitudinal observations on case V -7 and V-8 with the Arg-135-Trp mutation over a I-year span. Asevere visual field area loss from baseline was seen for both investigated targets (on average, 46% for the V4e target and 53% for the I4e target). These data confirm the peculiar clinical and functional severity of RP in this pedigree, demonstrating also a rate of progression much higher than expected based on Berson's45 data on the yearly progression rates determined on a large RP population, and also compared with Jacobson et a1' s25 findings on one patient with the Glu-64-ter mutation over a 7-year period. Longitudinal ERG observations also were available for case V -7. A yearly rate of ERG amplitude progression consistent with visual field data was demonstrated (on average, 54% for 0.5-Hz ERG and 50% for 3D-Hz ERG). Progression of retinal dysfunction also was

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documented by increases in peak times for both a- and b-waves. Also, these data differ substantially from the generally accepted progression rates for patients with Rp46 and from the patient with Glu-64-ter examined by Jacobson et a1. 25 Finally, also visual acuity appears to deteriorate greatly with disease duration, proportionally to the extent of foveal atrophy (Fig 3). Macular edema was never observed in this subset of patients. These observations are consistent with Farber et a1' s46 observations about patients with adRP with diffuse (type 1) retinopathy. In conclusion, our data confirm that the phenotype associated with the Arg-135-Trp mutation of the rhodopsin molecule is always severe, with only 3% to 4% of maximal ERG amplitude remaining as early as by 6 years of age. Later in the course of the disease, macular function also is severely affected, leaving only residual central vision by the fourth decade of life. Absence of intrafamilial and interfamilial variability associated to mutations at this codon further emphasizes the crucial role of this residue. In line with this observation, an unusually high progression rate of the disease was documented, suggesting that a mutation at this site is extremely deleterious to rod survival and causes a dramatically fast deterioration of retinal function as a whole. If the yearly progression rates observed in this study were assumed to be linear over time, maximal ERG responses could be predicted to be virtually nonrecordable in all patients by 20 years of age. It will be of interest to determine, in future longitudinal studies, whether different mutations eventually causing a common severe pattern yield a different clinical and functional evolution and entail a different susceptibility to be influenced by epigenetic factors (e.g., cigarette smoking, diet, psychophysical stress, and immunological factors). Longitudinal observations in this study were limited to a few patients, and therefore might not be extendable to all patients with this genotype. It will be the object of future investigations to verify whether all other patients show a similar rate of progression, and if this remains constant over the years.

Acknowledgments. Part of the molecular genetic analyses were performed by May al-Maghtheh under the supervision of Prof. Shomi S. Bhattacharya at the Department of Molecular Genetics, Institute of Ophthalmology (associated with Moorfields Eye Hospital), University of London, England.

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