Ocular Findings in a Form of Retinitis Pigmentosa with a Rhodopsin Gene Defecu*

OCULAR FINDINGS IN A FORM OF RETINITIS PIGMENTOSA WITH A RHODOPSIN GENE DEFECU*

BY Eliot L. Berson, MD

INTRODUCTION THE INCIDENCE OF RETINITIS PIGMENTOSA IS ESTIMATED TO BE ABOUT 1 in 3,500 births in the United States.",2 In the state of Maine, approx- imately 43% of cases are from families with an autosomal dominant mode of transmission, 20% are autosomal recessive, and 8% are X-linked; 23% are isolated cases with no family history, and 6% are undetermined (eg, adopted).2 Genetic heterogeneity is thought to exist within each hereditary pattern; for example, linkage studies have shown at least two genetic loci for the dominant and X-linked forms.3-8 Therefore, retinitis pigmentosa is a group ofdiseases caused by abnormal genes at various loci within the human genome. The symptoms and signs of retinitis pigmentosa are well known.9-2' Patients with this group of diseases characteristically develop night blindness and difficulty with mid-peripheral visual fields; as their condition progresses, they lose far-peripheral field and eventually central vision. Signs on ocular examination in more advanced stages include attenuated retinal arterioles, intraretinal bone spicule pigment around the periphery, waxy pallor of the optic discs, and vitreous abnormalities. Posterior subcapsular cataracts develop in many cases,22 and some patients show cystoid macular edema.2324 Refractive errors, including myopia and astigmatism, are common.2526 Patients have abnormal electroretinograms (ERGs),27 even in the early stages in the absence of visible abnormalities on fundus examination.28-31 Relatives with normal ERGs have not been observed to develop retinitis pigmentosa at a later time.32-34 Most pa-

*From the Berman-Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts. This research was supported in part by National Eye Institute grants EY00169 and EY02014 and grants from the National Retinitis Pigmentosa Foundation, Baltimore, Maryland, and the George Gund Foundation, Cleveland, Ohio.

TR. AM. OPHTH. Soc. vol. LXXXVIII, 1990 356 Berson tients with autosomal dominant retinitis pigmentosa retain some vision after age 60, while the majority of patients with autosomal recessive disease and X-linked disease become virtually blind by ages 45 to 60 and 30 to 45, respectively. Loss of photoreceptors as well as photoreceptors with shortened or absent outer segments have been observed in autopsy eyes from affected patients.35-46 No treatments are known for practically all types of retinitis pigmentosa except the rare forms associated with the Bassen-Kornzweig syndrome and Refsum's disease. Patients with the Bassen-Kornzweig syndrome have fat malabsorption, generalized retinal degeneration, diffuse neuromuscu lar disease with ataxia, acanthocytosis, and absence ofapolipoprotein B in plasma. 47-49 They have a deficiency ofchylomicrons and cannot efficiently absorb fat-soluble vitamins.50,51 Large doses ofvitamin A, administered in the early stages, have resulted in a return of dark adaptation thresholds and ERG responses to normal.52'53 Vitamin E and omega-3 fatty acids (ie, eicosapentaenoic acid and docosahexaenoic acid) have also been advo- cated to prevent progression of this retinal degeneration.54-57 Those with advanced stages have not responded to treatment53 and have extensive loss of photoreceptor cells.58'59 Patients with Refsum's disease have a peripheral neuropathy, ataxia, an elevated cerebrospinal fluid protein with a normal cell count, and retinitis pigmentosa. Anosmia, nerve deaf- ness, electrocardiographic changes, skeletal malformations, and ichthyo- sis occur in some cases.60,61 All have elevated serum phytanic acid due to a phytanic acid oxidase deficiency.6263 The pathogenesis appears to involve accumulation of exogenous phytanic acid in a variety of tissues, including the retinal pigment epithelium.64'65 Treatment with a low phy- tol-low phytanic acid diet (ie, excluding animal fats, milk products, and green leafy vegetables) has resulted in lowering of serum phytanic acid, improvement of motor nerve conduction velocity, alleviation of ataxia, lowering of the cerebrospinal fluid protein, and stabilization of the retinitis pigmentosa and hearing loss.61-63 Molecular genetic techniques have provided a new approach to finding biochemical abnormalities in retinitis pigmentosa as well as in other hereditary diseases. A series of deoxyribonucleic acid (DNA) probes linked to various regions of the human genome, if found to cosegregate with a form of retinitis pigmentosa, can be used to localize the region of a chromosome where the gene for that form is located. Recently, a gene responsible for autosomal dominant retinitis pigmentosa in a large family in Ireland was found to be linked to an anonymous polymorphic sequence named CRI-C17 within the long arm of human chromosome 3.3,66 The gene coding for rhodopsin was a likely candidate, as the rhodopsin gene is RP with Rhodopsin Gene Defect 357 also within the long arm ofchromosome 3,67,16 and rhodopsin is expressed in rod photoreceptors which are affected early in this disease.69'70 In addition, defects in related human cone opsin genes have been associated with cone photoreceptor degeneration.71'72 These findings prompted a search for an abnormality in the rhodopsin gene in the leukocyte DNA of patients with dominant retinitis pigmentosa. The search revealed an abnormal nucleotide sequence in the gene coding for rhodopsin (Fig 1) in 17 of 148 unrelated patients with autosomal dominant retinitis pigmentosa and in 0 of 102 unaffected individuals.5 This nucleotide base change was a cytosine to adenine transversion (ie, CCC to CAC) in codon 23, corresponding to a substitution ofhistidine for proline in the twenty-third amino acid of rhodopsin. In one large family studied, only clinically affected relatives showed this gene defect.5 These results, coupled with the fact that proline 23 is highly conserved among normal opsins (Table I),73 suggested that this point mutation affected a

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FIGURE 1 Representation of opsin in the rod outer segment membrane. Arrow designates the pre- dicted site of mutation in this protein in the 23rd amino acid from the amino terminus in patients with one form of dominant retinitis pigmentosa. Glycosylation sites (X) are designated for amino acid residues at positions 2 and 15. The site ofattachment ofopsin to ll-cts- retinal at lysine 296 to form rhodopsin (*) is located in the seventh transmembrane segment, separate from the site of this mutation. The carboxy terminal tail is thought to be anchored to the outer segment disc membrane by palmitate attached to cysteine 322 and cysteine 323, thereby forming the fourth loop on the cytoplasmic side. Shaded circles indicate amino acids that are invariate among vertebrate opsins. 358 Berson

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{,8 ffi; 2 ,,,c, c c c A A 8~~C RP with Rhodopsin Gene Defect 359 critical amino acid in rhodopsin and that this mutation could be the cause of this form of autosomal dominant retinitis pigmentosa.5 In the present study the findings on ocular examination ofthe 17 of 148 unrelated patients with dominant retinitis pigmentosa and this C to A transversion in codon 23 of the rhodopsin gene are described for the first time. Their findings are compared with those of the 131 unrelated patients with dominant retinitis pigmentosa without this mutation to show that differences exist, on average, between these two groups. In addition, 11 affected relatives of the 17 patients are presented to show that clinical heterogeneity exists even though these patients and their relatives have the same point mutation in the rhodopsin gene. Some mechanisms by which this mutation in rhodopsin could lead to photoreceptor cell death will be discussed. Some opportunities for future clinical and laboratory research in search of possible treatment will be considered.

METHODS

PATIENT SELECTION Clinical findings were reviewed from 148 patients, age 18 to 49 years, with autosomal dominant retinitis pigmentosa who had donated a blood specimen for molecular genetic studies of their leukocyte DNA. All 148 patients were from separate families, and they resided in the United States or Canada. Each patient was from a family with a dominant mode of transmission of retinitis pigmentosa over at least three consecutive generations. Seventeen of these patients had the C to A transversion in codon 23 of the rhodopsin gene, corresponding to a substitution of histidine for proline, while 131 of these patients did not carry this mutation.5 All 148 patients had retinal arteriolar attenuation, and the majority had intraretinal bone spicule pigmentation around the periphery. Ocular findings were also reviewed in 11 clinically affected relatives of4 ofthe 17 patients with the mutation. These relatives were selected because they had al- ready provided blood specimens for DNA analysis; these relatives had the same C to A transversion in codon 23 of the rhodopsin gene. Nucleotide sequences ofcodons 20 to 26 ofthe human rhodopsin gene, obtained from leukocyte DNA with methods previously described,5 are illustrated in Fig 2 for a normal individual (N79), a patient with dominant retinitis pigmentosa without this mutation (AD12), and 4 of the 17 patients with this mutation (AD160, AD133, AD87, and AD126). The normal individual and patient AD12 without the mutation show the normal74 sequence, while the four patients with the mutation show the C to A base change within codon 23 (ie, CCC to CAC) that results in a 360 Berson substitution of histidine for proline. Sequencing gels were initially used to evaluate a 558 base pair fragment of exon 1 of the rhodopsin gene, which had been amplified with the polymerase chain reaction, in 20 of the 148 patients, of whom 5 showed the mutation.5 Then, two oligomers (19mers) corresponding to the normal (5'-ACGCAGCCCCTTCGAGTAC-3') and mutant (5'-ACGCAGCCACTTCGAGTAC-3') sequences in codon 23 were synthesized. These oligomers, end-labeled with gamma 32p [adenosine 5'-triphosphate tetra (triethylammonium) salt], were individually hy- bridized with the amplified DNA from each of the 148 unrelated patients, as well as from 102 normal individuals as controls. Seventeen of the 148 patients, including the original 5 patients analyzed with sequencing gels, carried the C to A base change by this technique, whereas the 102 normal individuals did not show this mutation (P< 0.001), thereby excluding the possibility that the nucleotide change represented a DNA polymorphism

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FIGURE 2 Nucleotide sequence of codons 20 to 26 of the human rhodopsin gene derived from leukocyte DNA for a normal individual (N79), and from five representative patients with autosomal dominant retinitis pigmentosa included in this study and identified by their four nucleotide molecular genetic numbers AD12, AD160, AD133, AD87, and AD126. The G bases, namely, cytosine, thymine, adenine and guanine are designated by C, T, A, and for each patient. The normal and patient AD12 show the normal sequence while patients AD160, AD133, AD87, and AD126 are heterozygous for the C to A transversion within codon 23 (CCC to GAG). These DNA sequences were obtained from leukocyte DNA as described previously. RP with Rhodopsin Gene Defect 361 with no relationship to the disease. Nucleotide sequences of other exons of rhodopsin were also isolated from leukocyte DNA from some of the 17 affected patients and no other point mutations resulting in amino acid substitutions in rhodopsin were found.5 The 11 relatives with retinitis pigmentosa in the present study were evaluated either with sequencing gels or with the synthesized oligomers, and all showed the same C to A mutation in codon 23.

OCULAR EXAMINATION The 148 unrelated patients with dominant retinitis pigmentosa, including the 17 with the C to A transversion and the 131 without the C to A transversion in codon 23, completed a questionnaire with respect to age of onset of night blindness and age of onset of difficulty with side or peripheral vision and then were evaluated with respect to distance visual acuities, kinetic Goldmann visual fields, applanation tensions, dark adapted full-field ERGs, retinal acuities, slit-lamp appearance ofeach lens, and fundus appearance by ophthalmoscopy. Best corrected distance visual acuity was first measured for each eye with undilated pupils at a distance of 3.2 meters on a projected Snellen chart. Distance acuity was then measured for each eye on a retro-illuminated Ferris chart; the Ferris charts contained 5 Sloan letters of comparable difficulty on each line.75 Two different Ferris charts were used, one for each eye to avoid any possible biases due to memorizing the chart. After measuring distance acuity, kinetic visual fields were obtained with a V-4e white test light on a background of 31.5 apostilbs in the Goldmann perimeter. The test light was moved from nonseeing to seeing areas. The fields were plotted with a digitizing tablet and the total area for each eye was quantitated by a computer in degrees squared and expressed as an equivalent circular area or equivalent circular diameter.76 Applanation tensions were then measured in mm Hg for each eye at the Haag-Streit slit-lamp after topical anesthesia with proparacaine hydrochloride and topical application of fluorescein. After maximal dilation of the pupils with phenylephrine hydrochloride and cyclopentolate hydrochloride and dark adaptation for 45 minutes, full-field ERGs were recorded from each eye with a bipolar Burian-Allen contact lens electrode placed on the topically anesthetized cornea. Full- field ERGs were elicited in response to 0.5 Hertz (Hz) flashes of white light and then 30 Hz white light flashes presented in the Ganzfeld dome. Stimulus duration was 10 ,usec and stimulus luminance was 3.8 log foot- Lamberts (ft-L). Responses were differentially amplified at a gain ofup to 10,000 (-3 decibels at 2 Hz and 300 Hz), attenuated at 60 Hz with a notch filter (Q = 30) and for 30 Hz flickering stimuli, further amplified at a gain 362 Berson of up to 20 with a bandpass filter (Q = 16). Responses were summed by a computer (n = 64 for 0.5 Hz responses and n = 768 for 30 Hz responses). The computer contained an artifact reject buffer to eliminate eye move- ments and blink voltages exceeding an adjustable minimum of + 50 microvolts (RxV) for the 0.5 Hz condition and + 2.5 ,uV for the 30 Hz condition. Under these test conditions responses to 0.5 Hz flashes could be detected iftheir amplitudes were - 1.0 RuV and to 30 Hz flicker iftheir amplitudes were - 0.05 RiV.76 Responses were quantified with respect to peak-to-peak amplitudes for the mixed cone-rod responses to 0.5 Hz white flashes and the cone-isolated responses to 30 Hz white flashes and with respect to cone b-wave implicit times (ie, stimulus onset to the major cornea-positive peak of the 30 Hz white flashes). After ERG testing, retinal acuity was obtained for each eye on a Snellen equivalent number chart with the Guyton-Minkowski potential acuity meter (ie, PAM TM). 77,78 Slit-lamp examination was done to determine presence or absence of a central posterior subcapsular cataract in each eye. Ophthalmoscopic examination was then performed on each eye to determine whether or not cystoid macular edema could be seen and whether intraretinal bone spicule pigment was present in the periphery in all four quadrants. The 11 relatives also completed a questionnaire with respect to age of onset of night blindness and age of onset of difficulty with side or peripheral vision and were then evaluated with respect to best corrected Snellen visual acuities, kinetic visual fields with a V-4e white test light, applanation tensions, final dark adapted rod psychophysical thresholds, dark adapted full-field ERGs, slit-lamp appearance of the lens, presence or absence ofcystoid macular edema, and presence or absence ofintraretinal bone spicule pigment in all four quadrants around the periphery. Dark adapted rod psychophysical thresholds were measured with the Gold- mann Weekers adaptometer after 45 minutes of dark adaptation to an 110 white test light fixated centrally or 70 above fixation. Full-field ERGs were elicited to single flashes (0.5 Hz) of dim blue light (1.2 log ft-L, X < 470 ni), single flashes (0.5 Hz) of white light (3.8 log ft-L), and 30 Hz white flicker (3.8 log ft-L) without computer averaging for responses greater than 10 ,uV and with computer averaging, as described above, ifresponses were less than 10 RV.

STATISTICAL ANALYSIS The data based on ocular examinations for the 148 patients with dominant retinitis pigmentosa were coded by one person and checked by another; data were then keypunched, verified, and stored on a magnetic tape for RP with Rhodopsin Gene Defect 363 data processing. All data were then validated for errors and inconsisten- cies and corrected appropriately. Comparisons were done between the group of 17 unrelated patients with this mutation and the group of 131 unrelated patients without this mutation. To minimize intra-individual variability, all 148 patients were examined twice within 6 weeks by the author, and an average for each eye across both visits was used for each test parameter. Since the distribution of Snellen distance visual acuities, retinal visual acuities, visual field equivalent circular areas, and ERG amplitudes were skewed, the data were transformed using the loge scale to better approximate a normal distribution for each parameter for purposes ofanalysis. Data for continu- ous variables such as best corrected visual acuity or spherical refractive error were analyzed using t-tests for univariate analyses.79 Discrete variables such as presence or absence ofposterior subcapsular cataract in both eyes or presence or absence of bone spicule pigment in all 4 quadrants of the fundus periphery in both eyes were analyzed with the Yates-corrected chi-square test for 2 x 2 tables or Fisher's exact test.79 Age of onset of night blindness and age of onset of difficulty with side vision were evaluated by using the log rank life table method ofanalysis; patients who were not yet night blind or who had not yet reported field loss were treated as censored observations with length of follow-up equal to their current age. Multiple regression analysis was employed using group membership, age, and sex as the independent variables and selected visual measurements as the dependent variables. This allowed assessment ofdifferences between the two groups for selected visual parameters after correcting for age and sex.8081 The 11 affected relatives were also examined by the author. The number of affected relatives on file was considered too small for statistical analysis. Their clinical findings are presented to provide additional data on the range of ocular abnormalities seen in patients with this mutation and to show examples of intrafamilial clinical heterogeneity. RESULTS Findings on history for the 17 patients from separate families with autosomal dominant retinitis pigmentosa and the C to A transversion in codon 23 of the rhodopsin gene are summarized in Table II. There was variability in the age of onset of night blindness reported by these patients. For example, one patient reported onset of night blindness near birth while another denied any symptoms ofnight blindness at age 38. Most did not report difficulty with side vision until after age 20, but one patient reported onset at age 15, while another denied any loss of side vision at age 45. 364 Berson

Findings on ocular examination for the 17 unrelated patients with this mutation are described in Table III. These patients showed best corrected Snellen distance visual acuities in the range of 20/20 to 20/62, based on an average of two measurements obtained within 6 weeks. Ferris distance acuities were consistent with the Snellen acuities for each patient. Retinal visual acuities on a Snellen equivalent number chart were also comparable to the distance acuity measurements for each patient. Eight of 17 (47%) had 1.0 or more diopters of astigmatism in at least one eye. Seven of 17 (41%) showed central posterior subcapsular cataracts in both eyes. Intraretinal bone spicule pigment was present in all four quadrants of both eyes in 13 of 17 (76%). Findings were comparable in both eyes of a given patient in practically all cases. Interfamilial clinical heterogeneity was evident, as some older patients reported better visual acuities than younger patients; some older patients had clear lenses and absence of bone spicule pigment in all four quadrants, while younger patients had central posterior subcapsular cataracts and extensive pigment around the peripheral fundus. Visual field areas to a V-4e white test light and ERG amplitudes and implicit times for the 17 patients with this mutation are presented in

TABLE II: FINDINGS ON HISTORY IN AFFECTED PATIENTS AGE OF ONSET AGE OF ONSET PATIENT FAMILY OF NIGHT OF DIFFICULTY NO NO AGE* SEX BLINDNESS WITH SIDE VISION 1 5375 23 M Nonet Nonet 2 5938 26 F 0O 23 3 5727 29 F 25 26 4 1566 29 F 20 None 5 7053 32 M 14 28 6 6038 33 M 5 23 7 6994 33 F 12 25 8 5968 34 M 15 24 9 6690 38 M 17 23 10 2317 38 F None None 11 6281 39 M 3 34 12 6734 42 F 38 39 13 6653 43 F 6 15 14 5998 43 F 34 39 15 6174 44 M 31 32 16 5767 45 F 12 None 17 5850 52 F 4 20 *Age at time of visit. tNone designates patient has not yet experienced symptom. t0 designates onset of symptom near birth. RP with Rhodopsin Gene Defect 365

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+ zSzo k- 4 -4 z z RP with Rhodopsin Gene Defect 367 Table IV. Visual field areas, expressed as equivalent circular areas or diameters, ranged from normal in 4 of 17 (ie, > 11,399 degrees squared or 2 120 degree equivalent circular diameter) to very constricted in 2 of 17 (s< 334 degrees squared or - 210 diameter). Visual field areas were comparable in both eyes of each patient. Some older patients retained larger visual field areas than younger patients. ERG amplitudes were abnormal to 0.5 Hz light in all and 30 Hz flicker in most cases; for the most part ERG amplitudes were comparable in both eyes. Older patients in some instances showed larger amplitudes than younger patients. Cone ERG b-wave implicit times were delayed in practically all cases. Mean values for Snellen distance acuity, Ferris visual acuity, retinal acuity, spherical and cylindric refractive errors, applanation tensions, visual field areas, and ERG amplitudes and implicit times for the patients with this mutation (group I) versus the patients without this mutation (group II) are presented in Table V. The mean age (±+ SD) ofgroup I (n = 17) was 36.6 ± 7.7 years and ofgroup II (n = 131) was 32.1 + 8.3 years (P = 0.043). Although group I was older on average, they retained significantly better distance and retinal acuity. ERG amplitudes, both to 0.5 Hz white light and 30 Hz white flicker, were on average about three times larger in group I than in group II, while cone b-wave implicit times to 30 Hz white flicker were not significantly different between the two groups. Mean spherical and cylindric refractive errors and mean applanation tensions were not significantly different between the two groups. Mean visual field area was slightly larger in group I (3463 degrees squared or 66 degree equivalent circular diameter) than in group II (2719 degrees squared or 59 degree equivalent circular diameter), but the groups were not significantly different from each other. Sex ratios between group I (41% males, 59% females) and group II (54% males, 46% females) also were not significantly different from each other. A life table analysis comparing the two groups with respect to age of onset ofnight blindness by history showed that the median age ofonset of night blindness was 14 years for group I and 13 years for group II (P = NS by log rank test). In contrast, a life table analysis revealed a significant difference between the two groups with respect to age of onset of difficulty with side vision by history; the median age ofonset ofdifficulty with side vision was 26 years for group I and 21 years for group II (P = 0.037 by log rank test). Centrai posterior subcapsular cataracts in both eyes were present in 7 of 17 (41%) in group I and 72 of 125 (58%) in group II. Intraretinal bone spicule pigment was present in all four quadrants in both eyes in 13 of 17 (76%) in group I and 106 of 131 (81%) in group II). Cystoid macular edema 368 Berson

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FIGURE 3 Families 5850, 1566, 5375, and 5767 with autosomal dominant retinitis pigmentosa with a C to A transversion in codon 23 of the rhodopsin gene. Solid symbols, affected individuals by ocular examination; open symbols, unaffected individuals by ocular examination or by history; slashed symbols, deceased individuals. Oblique arrows indicate 4 of the 17 unrelated patients with this mutation whose clinical findings are presented in Tables II, III, and IV. Sequencing gels from the four affected patients with this mutation (ie, patients 17, 4, 1, and 16) are, respectively, designated by molecular genetic numbers AD160, AD133, AD87, and AD126 in Fig 2. All affected relatives for whom ocular examinations, ERGs, and DNA analyses were available are included in Tables VI, VII, and VII. 372 Berson

TABLE VI: FINDINGS ON HISTORY IN AFFECTED RELATIVES AGE OF ONSET AGE OF ONSET PATIENT FAMILY OF NIGHT OF DIFFICULTY NO NO PEDIGREE* AGE SEX BLINDNESS WITH SIDE VISION 18 5850 III-3 24 F Nonet Nonet 19 5850 III-1 29 F None None 20 5850 II-7 50 F None None 21 1566 III-5 25 F None None 22 1566 III-i 37 M None None 23 1566 II-1 63 M 16 40 24 5375 III-i 28 M Ot None 25 5375 II-4 48 F None None 26 5375 II-i 53 M 0 40 27 5767 III-4 54 M 30 None 28 5767 II-Il 73 F 15 40 *Relative position in pedigree for each family in Fig 3. tPatient has not yet experienced symptom. tOnset of symptom near birth. For example, in family 5850 three members, ages 24, 29, and 50, denied night blindness, while their relative (patient 17) reported night blindness at age 4 (Table II). In family 5767, patient 27 had not noticed loss of side vision at age 54, while patient 28 noticed loss of side vision at age 40. In family 5767, visual acuities ranged from 20/400 in patient 27, age 54, to 20/80 in patient 28, age 73 (Table VII). Bone spicule pigment was present in all four quadrants ofboth eyes in some members and not in others in all four families; older relatives without pigment and younger relatives with pigment were observed in families 5850 and 5375 (Table VII). Final dark adaptation thresholds, visual field areas, and full-field ERG amplitudes for the 11 relatives with this mutation are presented in Table VIII. The relatives in family 5850 had a minimal (ie, 0.3 log unit) elevation of final dark adapted rod thresholds consistent with their denial of any history ofnight blindness. Visual field areas ranged from normal in 6 of 11 relatives to very reduced in 2 of 11 (- 296 degrees squared or - 200 diameter). Intrafamilial variability was seen between family members; for example, in family 5375, patient 25, age 48, retained more field area than patient 24, age 28 (Table VIII). ERG amplitudes were reduced for all patients, to 0.5 Hz light and for most to 30 Hz flicker. Amplitudes varied widely with older relatives in some cases retaining larger amplitudes than younger relatives. Cone ERG implicit times to 30 Hz flicker varied from normal to substantially delayed in these relatives (Table VIII). Intrafamilial variability can also be seen in the ERG responses in family 5850 (Fig 4). Full-field ERG responses are illustrated from a normal member without this mutation (age 28) and from two clinically affected RP with Rhodopsin Gene Defect 373

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FIGURE 4 Full-field ERGs from an unaffected patient (age 28), two affected nieces (ages 24 and 29), and two affected aunts (ages 50 and 52) in family 5850 with autosomal dominant retinitis pigmentosa. Stimulus onset is the vertical hatched lines for the left and middle columns and vertical lines for the right column. Two or three consecutive sweeps are superimposed. Cornea positivity is an upward deflection. Oblique arrows in the middle column designate delayed rod-dominated peaks. Horizontal arrows in the right column designate cone b-wave implicit times, ie, time interval between stimulus flash and corresponding cornea-positive peaks. Calibration symbol (lower right) designates 50 ms horizontally and 100 p.V vertically. RP with Rhodopsin Gene Defect 377 siblings (patients 18 and 19) and two clinically affected aunts (patients 20 and 17) with this mutation. Rod responses to dim blue light are illustrated in the left column, mixed cone and rod responses to single flashes ofwhite light are illustrated in the middle column, and cone-isolated responses to 30 Hz white flickering light in the right column. The normal member has a normal ocular examination and also shows normal ERGs. The two affected siblings have markedly reduced rod responses to dim blue light and a splitting of the responses to white light into an early cone-dominated peak, and a delayed rod-dominated peak of reduced amplitude (oblique arrows, Fig 4). The splitting occurs as a consequence ofrelatively normal cone peak response times but substantially delayed rod peak response times. In contrast, the response ofthe 28-year-old normal member shows a cone and rod response to white light in which the two peaks cannot be distinguished. The cone response to 30 Hz white flicker of the 24-year-old affected sibling is normal in amplitude, while that of the 29- year-old affected sibling with more advanced disease is slightly reduced in amplitude with borderline delayed b-wave peak implicit times (horizontal arrows, Fig 4). The ERG findings in the two affected siblings are consistent with an early predominant involvement of rods in this form of retinitis pigmentosa as might be expected from a defect in rod-specific opsin. In the more advanced stage ofdisease, loss ofcone and rod function occurs as seen in the profoundly reduced responses to all three stimulus conditions for the 52-year-old affected aunt. Intrafamilial variability is illustrated in the 50-year-old aunt who shows predominant loss of rod function, but nevertheless retains responses to 0.5 Hz white light and to 30 Hz white flicker that are comparable to those ofher 24- and 29-year-old nieces and larger than those ofher 52-year-old sister. Review ofrecords of this 52-year-old patient indicated that she also had profoundly reduced (ie, < 10 ,uV) ERG responses at age 48, so that the differences in ERG amplitudes ofthe two oldest patients in this family did not depend on the ages of these patients at the time of testing. Representative fundus photographs from four patients are shown in Fig 5 to illustrate the variability that exists with respect to the amount of visible bone spicule pigment in patients with this mutation. Patient 6 (fundus photograph A), age 33, has substantially more bone spicule pigment than patient 16 (fundus photograph B), age 45. Patient 25 (fundus photograph C), age 48, and her brother, patient 26 (fundus photograph D), age 53, also show differences in the appearance of their fundi; review of records indicated that one sibling had pigment and one did not have pigment even when these siblings were evaluated at the same age. Comparison of patient 25 and patient 6 also shows that an older patient 378 Berson

FIGURE 5 Representative fundus photographs from four patients with autosomal dominant retinitis pigmentosa with a C to A transversion in codon 23 ofthe rhodopsin gene to show variability with respect to the extent ofintraretinal bone spicule pigmentation among patients with the same gene defect. from one family can have less bone spicule pigment than a younger patient from another family.

DISCUSSION The present study shows that 17 unrelated patients with autosomal dominant retinitis pigmentosa with a C to A transversion in codon 23 of the rhodopsin gene (corresponding to a substitution ofhistidine for proline in amino acid 23 of rhodopsin) have, on average, significantly better visual function as monitored by visual acuity and ERG amplitude than 131 unrelated patients with autosomal dominant retinitis pigmentosa without this mutation. Although interfamilial variability exists, patients with this RP with Rhodopsin Gene Defect 379 mutation retained, on average, seven letters more on the Ferris charts and two- to threefold larger ERG responses than those without this mutation. These differences were observed even though the patients with this mutation were on average 4.5 years older than those without this mutation. These data, based on findings at initial visits, raise two possi- bilities to explain the apparent disparity between these two groups with respect to loss of function. The first is that the time of onset of degeneration is later, on average, in the group with this mutation than in the group without this mutation, but that both groups have a similar rate ofdegener- ation; the second possibility is that the rate of degeneration is slower, on average, in the group with this mutation than in the group without this mutation. A prospective longitudinal study of subsets of patients with or without this mutation would help to explain this disparity. A previous prospective study of the natural course of retinitis pigmentosa indicated that patients lost, on average, 16% of remaining full-field ERG amplitude per year to a 0.5 Hz white light stimulus. 76 Based on this exponential rate of decline, an average patient with this mutation, who had an initial ERG amplitude of 14.4 ,uV (Table V), would be expected to take 33 years to reach 0.05 ,uV (ie, virtual blindness) while those without this mutation with an initial ERG amplitude of 4.9 ,uV (Table V) would be expected to take 27 years to reach 0.05 ,uV. Since the average ages of patients with this mutation and without this mutation were 36.6 years and 32.1 years, respectively, it can be estimated that patients in the group with this mutation would retain vision, on average, to about age 70 (ie, 33 + 36.6) while those without this mutation would retain vision to about age 59 (ie, 27 ± 32.1). Ifthe rate ofdecline in retinal function is slower in patients with this mutation coinpared to those without this mutation, then the differences between these groups could be even larger. Again, a long- term, prospective, longitudinal study would help to confirm this prediction. The wide range of time of onset of night blindness by history in the patients with this mutation would seem to preclude subtyping dominant retinitis pigmentosa into a type with early onset night blindness and a type with late onset night blindness, as suggested previously.82-84 Sim- ilarly, some patients showed normal cone ERG b-wave implicit times, while others were delayed, suggesting that families cannot be separated by this criterion into a dominant form with complete penetrance and a dominant form with reduced penetrance.31-3 The results from molecular genetic findings confirm previous clinical impressions that more than one type of dominant disease exists,31-3382-84 but the wide range of clinical findings in the patients with this mutation suggests that parameters of the 380 Berson ocular examination described in this study cannot be used precisely in any individual to assign a patient into one or another type. It is likely that the 131 patients without this mutation will be subdivided once other specific gene defects are discovered; given the predominant loss of rod function in these patients, it could be anticipated that other mutations in the rhodopsin gene will be found in some of these patients. Clinical comparisons between subgroups will be of interest if additional etiologi- cal mutations in the rhodopsin gene are found. Since the proline at position 23 is invariate among normal vertebrate and invertebrate opsins (Table I), it would be expected that this highly conserved amino acid, if altered radically, as would occur with the substitution of a charged amino acid histidine for a nonpolar hydrophobic proline in the amino-terminus, would lead to a dysfunctional or absent rhodopsin molecule within the rod outer segment membrane.5 The ocular findings in these patients are consistent with this idea, as a predominant loss of rod function is typically seen in the ERGs of patients with early stages of this form of retinitis pigmentosa.30 Rod responses are not only reduced in amplitude but are also delayed in implicit time, a phenome- non that can be simulated in normal subjects by interposing a neutral density filter between the stimulus flash and the eye (ie, simulation in normals of reduced amount of visual pigment).30,85,86 The reduced and delayed rod ERGs in the patients are therefore compatible with a reduction in the amount of rhodopsin and/or a dysfunctional rhodopsin in remaining rods in the early stages.30 Rod photoreceptors undergo daily renewal of outer segments at their base and and phagocytosis of their distal (ie, apical) outer segment tips as schematically presented in Fig 6, so that rod outer segments are com- pletely renewed on average about every ten days. 87-91 Rhodopsin is glycosylated at asparagine residues located at positions 2 and 15 in the amino-terminus (Fig 1).92 This glycosylation is thought to be involved in rod outer segment disc assembly, since inhibitors ofglycosylation, such as tunicamycin, can interrupt the assembly of outer segment discs.93,94 Therefore, an abnormal amino-terminus could lead to an abnormality of disc assembly with shortening of rod outer segments. The amino-terminus of rhodopsin faces the intradiscal lumen (Fig 6) for those molecules of rhodopsin in the outer segment disc membrane95'96; an abnormality in this amino-terminus could possibly modify transport of this end of the molecule across disc membranes. The amino-terminus of rhodopsin faces the extracellular space for those molecules of rhodopsin in the plasma membrane; an abnormality in the amino-terminus could, therefore, possibly affect rod photoreceptor-pigment epithelial cell interactions. Since RP with Rhodopsin Gene Defect 381

OUTER SEGMENT

INNER POST GOLGI VESCLES C SEGMENT GOLGI B ROUGH ENDOPLASMIC RETICUWM A

NUCLEUS

SYNAPTIC BODY

FIGURE 6 A schematic representation of a normal human rod photoreceptor to illustrate steps in delivery of opsin to outer segments. Opsin is synthesized and glycosylated in ribosomes of the rough endoplasmic reticulum (A), further modified in the Golgi complex (B), and transported toward the cell surface by post-Golgi vesicles (C) where it is thought to be incorporated into the plasma membrane at the level of the connecting cilium. Transfer of opsin from inner to outer segment is not fully understood. New disc formation (D) appears to involve evagination of the plasma membrane of the distal connecting cilium. Opsin appears to acquire vitamin A (ie, 11-cis-retinal) at the base ofthe outer segment, and probably in the inner segment as well, and this confers light sensitivity on this protein now called rhodopsin. 382 Berson proline sites have been considered important in defining the shape ofrhodopsin,92 it is also possible that an abnormal bending of the amino- teminus as a consequence ofa histidine substitution for proline could lead to a conformational change in rhodopsin with a deleterious effect on rod function and viability. Abnormal rod ERG diurnal rhythms have been previously reported in light-entrained young adult patients from family 1566 with dominant retinitis pigmentosa with this mutation; these patients had abnormal reductions in rod ERG sensitivity 1.5 and 8 hours after light onset.97 The abnormal reduction in rod sensitivity raised the possibility that these patients lost abnormally large fractions of rod outer segments and that rods were slow to renew their pre-light onset outer segment length. These abnormal rod ERG diurnal rhythms are consistent with an abnormality in rod renewal that could be occurring as a result of this gene defect. The gene defect can be associated with a complete loss of rods, as autopsy eyes from a patient, age 68, from this family (ie, patient 1-2 in family 1566 in Fig 3) showed complete loss of rods and a few remaining foveal cones with shortened and disorganized outer segments.38 The precise mechanism by which a mutation in a gene thought to be expressed exclusively in rods can lead to widespread degeneration of both rods and cones in these patients remains to be defined. All patients studied so far with this mutation, now designated as rhodopsin, Pro-23-His, have shown evidence ofretinitis pigmentosa based on ocular examination and ERG and no unaffected individual has shown this mutation5; this supports the idea that the diagnosis ofthis form of retinitis pigmentosa can be made from a sample ofperipheral blood. In view ofthe clinical heterogeneity that exists among those with this mutation, patients identified by molecular genetic analysis should have an ocular examination and ERG to determine the stage of their condition. Since some patients with this mutation are asymptomatic in adulthood with minimal, if any, bone spicule pigment, asymptomatic relatives of patients with this mutation should have their leukocyte DNA evaluated for this mutation or have an ERG before concluding that they do not have this disease. Leukocyte DNA analysis should be considered not only for families with a dominant mode of transmission over three consecutive generations but also for families with transmission over two generations or for isolated cases with presumably normal parents, as some ofthese patients may have this mutation, thereby establishing that the mode ofinheritance is autosomal dominant. Patients identified with this mutation would know that they have a 50% chance of having an affected child with each childbirth. RP with Rhodopsin Gene Defect 383

Knowledge of the precise gene defect in a form of dominant retinitis pigmentosa provides new opportunities to understand the pathogenesis of the disease and seek means to modify its course. It may be possible to inject mutant gene constructs into mouse eggs and create transgenic mice with this human mutation.98-100 These mice in turn could be studied in an effort to define the pathogenesis and search for treatments. The mutant rhodopsin molecule can be synthesized in the laboratory10l and its expression studied in a variety of in vitro systems. 102103 An antibody produced to the mutant rhodopsin could be applied to autopsy eyes from patients with this mutation to determine the amount and distribution of mutant rhodopsin in remaining rod photoreceptors to help clarify pathogenesis. Light deprivation'04105 and light stress'06 as they may affect photoreceptors with this mutant rhodopsin can also be studied. The reason for the apparently slow rate of progression, on average, of affected patients with this mutation remains to be clarified. It remains to be proven whether a mutant rhodopsin protein is made and, if made, whether it interferes with photoreceptor function at the level ofthe inner or outer segment. The mutation is in the amino-terminus rather than near the 11-cis-retinal binding site or the binding sites for transducin in the cytoplasmic loops (Fig 1); this raises the possibility that the defect only partially interferes with the function ofrhodopsin, thereby accounting for the slow course. The wide range of clinical findings described in the present report for the 17 affected patients and their 11 relatives, with older patients sometimes having less advanced disease than younger patients, shows that clinical heterogeneity can exist in patients with retinitis pigmentosa with the same point mutation in the rhodopsin gene. This wide range of findings suggests that some factor other than the defect in the gene itself is involved in the expression of this disease. As more patients with this rhodopsin gene defect are identified, risk factor analyses could be performed on patients with varying severity of disease in search of aggravat- ing or ameliorating factors, with possible implications for treatment.

SUMMARY Ocular findings are presented in 17 unrelated patients with a form of autosomal dominant retinitis pigmentosa and the same C to A transversion in codon 23 of the rhodopsin gene. These patients (mean age, 36.6 years) had, on average, significantly better visual acuity and larger ERG amplitudes than 131 unrelated patients (mean age, 32.1 years) with autosomal dominant retinitis pigmentosa without this mutation. These 17 384 Bersoln patients from separate families as well as 11 relatives with the mutation from 4 of these families showed interfamilial and intrafamilial variability with respect to severity oftheir ocular disease. This clinical heterogeneity among patients with the same mutation, with older patients sometimes showing less loss of visual function and less intraretinal bone spicule pigment than younger patients, suggests that some factor other than the gene defect itself is involved in the expression of this condition. This form of retinitis pigmentosa can now be detected by testing leukocyte DNA from peripheral blood. Patients so identified should have an ocular examination to determine the extent of their disease in view of the clinical heterogeneity that exists among patients with this mutation. Some mechanisms by which this mutation in the rhodopsin gene could lead to photoreceptor cell death are discussed. Opportunities for future clinical and laboratory research in search of possible treatments are considered.

ACKNOWLEDGMENTS The author thanks Drs Thaddeus P. Dryja, Bernard Rosner, Michael A. Sandberg, and Elias Reichel for their advice. The author also wishes to thank Carol Weigel-DiFranco, Terri L. McGee, Lauri B. Hahn, Glenn S. Cowley, Catherine LaDow, Amanda M. Bruce, Michelle Martinez, Jen- nifer Morrow, and Basil Pawlyk for their technical assistance. Kathleen Tauson typed the manuscript.

REFERENCES 1. Boughman JA, Conneally PM, Nance WE: Population genetic studies of retinitis pigmentosa. Am J Hum Genet 1980; 32:223-235. 2. Bunker CH, Berson EL, Bromley WC, et al: Prevalence of retinitis pigmentosa in Maine. Am J Ophthalmol 1984; 97:357-365. 3. McWilliam P, Farrar GJ, Kenna P, et al: Autosomal dominant retinitis pigmentosa (ADRP): Localization of an ADRP gene to the long arm of chromosome 3. Genomics 1989; 5:619-622. 4. Inglehearn CF, Jay M, Lester DH, et al: The evidence for linkage between late onset autosomal dominant retinitis pigmentosa and chromosome 3 locus D3547 (C17): Evi- dence for genetic heterogeneity. Genomics 1990; 6:168-173. 5. Dryja TP, McGee T, Reichel E, et al: A point mutation of the rhodopsin gene in one form of retinitis pigmentosa. Nature 1990; 343:364-366. 6. Bhattacharya SS, Wright AF, Clayton JF, et al: Close genetic linkage between X-linked retinitis pigmentosa and a restriction fragment length polymorphism identified by recombinant DNA probe L1.28. Nature 1984; 309:253-255. 7. Musarella MA, Burghes A, Anson-Cartwright L, et al: Localization of the gene for X-linked recessive type of retinitis pigmentosa (XLRP) to Xp2l by linkage analysis. Am J Hum Genet 1988; 43:484-494. 8. Chen JD, Halliday F, Keith G, et al: Linkage heterogeneity between X-linked retinitis pigmentosa and a map of 10 RFLP loci. Am J Hum Genet 1989; 45:401-411. RP withi Rhodopsin Gence Defect 385

9. Donders FC: Bertrage zur pathologischen Anatomie des Auges. Albrecht von Graefes Arch Klin Exp Ophthalmol 1855; 2:106-118, 1857; 1:139-165. 10. Leber T: Die Pigmentdegeneration der Netzhaut und mit ihr verwandte Erkrankun- gen, in Graefe Saemisch-Hess, Handbuch der Gesemten Augenheilkunde, ed 2, Vol 7. Leipzig, Germany, W Engelmann, 1916, pp 1076-1225. 11. Bell J: Retinitis pigmentosa and allied diseases of the eye, in Pearson K (ed): Treasury of Human Inheritance, Vol 2. London, Cambridge University Press, 1922. 12. Franceschetti A, Fran,ois J, Babel J: Les Heredodegenerescences Chorioretiniennes. Paris, Masson & Cie, 1963. 13. Duke-Elder S, Dobrie JH: System ofophthalmology, in Duke-Elder S (ed): Diseases of the Retina, Vol X. St Louis, CV Mosby, 1967, pp 574-622. 14. Merin S, Auerbach E: Retinitis pigmentosa. Surv Ophthalmol 1976; 20:203-346. 15. Deutman AF: Rod cone dystrophy: Primary hereditary, pigmentary retinopathy, retinitis pigmentosa, in Krill AE, Archer DB (eds): Krill's Hereditary Retinal and Cho- roidal Diseases: Clinical Characteristics, Vol II, Hagerstown, Harper & Row, 1977, pp 479-576. 16. Marmor MF, Aguirre G, Arden G, et al: Retinitis pigmentosa: A symposium on terminology and methods of examination. Ophthalmology 1983; 90:126-131. 17. Pruett RC: Retinitis pigmentosa: Clinical observations and correlations. Trans Am Ophthalmol Soc 1983; 81:693-735. 18. Heckenlively JR: RP cone-rod degeneration. Trans Am Ophthalmol Soc 1987; 85:438- 470. 19. Pagon RA: Retinitis pigmentosa. Surv Ophthalmol 1988; 33:137-177. 20. Heckenlively JR: Retinitis Pigmentosa. Philadelphia, JB Lippincott, 1988. 21. Newsome DA: Retinitis pigmentosa, Usher's syndrome, and other pigmentary reti- nopathies, in Newsome DA (ed): Retinal Dystrophies and Degenerations. New York, Raven Press, 1988, pp 161-194. 22. Heckenlively JR: The frequency of posterior subcapsular cataract in the hereditary retinal degenerations. Am J Ophthalmol 1982; 93:733-738. 23. Fetkenhour CL, Choromokos E, Weinstein J, et al: Cystoid macular edema in retinitis pigmentosa. Trans Am Acad Ophthalmol Otolaryngol 1977; 83:515-521. 24. Fishman GA, Maggiero JM, Fishman M: Foveal lesions seen in retinitis pigmentosa. Arch Ophthalmol 1977; 95:1993-1996. 25. Sieving PA, Fishman GA: Refractive errors of retinitis pigmentosa patients. Br J Ophthalmol 1978; 52:625-633. 26. Berson EL, Rosner B, Simonoff EA: Risk factors for genetic typing and detection in retinitis pigmentosa. Am J Ophthalmol 1980; 89:763-775. 27. Karpe G: The basis of clinical electroretinography. Acta Ophthalmol (Suppl) 1945; 23:84. 28. Goodman G, Gunkel RD: Familial electroretinographic and adaptometric studies in retinitis pigmentosa. Am J Ophthalmol 1958; 46:142-178. 29. Gouras P, Carr RE: Electrophysiological studies in early retinitis pigmentosa. Arch Ophthalmol 1964; 72:104-110. 30. Berson EL, Gouras P, Gunkel RD: Rod responses in retinitis pigmentosa, dominantly inherited. Arch Ophthalmol 1968; 80:58-67. 31. Berson EL, Simonoff EA: Dominant retinitis pigmentosa with reduced penetrance: Further studies of the electroretinogram. Arch Ophthalmol 1979; 97:1286-1291. 32. Berson EL: Retinitis pigmentosa, the electroretinogram, and Mendel's laws. Trans Pa Acad Ophthalmol Otolaryngol 1973; 26:109-113. 33. Retinitis pigmentosa and allied diseases: Electrophysiologic findings. Trans Am Acad Ophthalmol Otolaryngol 1976; 81:659-666. 34. Electroretinographic findings in retinitis pigmentosa. JpnJ Ophthalmol 1987; 31:327-348. 35. Verhoeff FH: Microscopic observations in a case of retinitis pigmentosa. Arch Oph- thalmol 1931; 5:392-407. 386 Berson

36. Cogan DG: Symposium: Primary chorioretinal aberrations with night blindness pathology. Trans Am Acad Ophthalmol Otolaryngol 1950; 54:629-661. 37. Mizuno K, Nishida S: Electron microscopic studies of human retinitis pigmentosa. Part I. Two cases ofadvanced retinitis pigmentosa. Am i Ophthalmol 1967; 63:791-803. 38. Kolb H, Gouras P: Electron microscopic observations of human retinitis pigmentosa dominantly inherited. Invest Ophthalmol Vis Sci 1974; 13:487498. 39. Szamier RB, Berson EL: Retinal ultrastructure in advanced retinitis pigmentosa. Invest Ophthalmol Vis Sci 1977; 16:947-962. 40. Szamier RB, Berson EL, Klein R, et al: Sex-linked retinitis pigmentosa: Ultrastructure of photoreceptors and pigment epithelium. Invest Ophthalmol Vis Sci 1979; 18:145- 160. 41. Meyer KT, Heckenlively JR, Spitznas M, et al: Dominant retinitis pigmentosa: A clinicopathologic correlation. Ophthalmology 1982; 89:1414-1424. 42. Santos-Anderson RM, Tso MOM, Fishman GA: A histopathologic study of retinitis pigmentosa. Ophthalmic Paediatr Genet 1982; 1:151-168. 43. Gartner S, Henkind P: Pathology of retinitis pigmentosa. Ophthalmology 1982; 89: 1425-1432. 44. Bunt-Milam AH,Kalina RE, Pagon RA: Clinical-ultrastructural study ofretinal dystrophy. Invest Ophthalmol Vis Sci 1983; 24:458-469. 45. Rodrigues MM, Wiggert B, Hackett J, et al: Dominantly inherited retinitis pigmentosa: Ultrastructure and biochemical analysis. Ophthalmology 1985; 92:1165-1172. 46. Flannery JG, Farber DB, Bird AC, et al: Degenerative changes in a retina affected with autosomal dominant retinitis pigmentosa. Invest Ophthalmol Vis Sci 1989; 30/2: 191-211. 47. Bassen FA, Kornzweig AL: Malformation of the erythrocytes in a case of atypical retinitis pigmentosa. Blood 1950; 5:381-387. 48. Jampel RS, Falls HF: Atypical retinitis pigmentosa, acanthocytosis, and heredodegen- erative neuromuscular disease. Arch Ophthalmol 1958; 59:818-820. 49. Salt HB, Wolff OH, Lloyd JK, et al: On having no betalipoprotein: A syndrome comprising a-beta-lipoproteinemia, acanthocytosis and steatorrhea. Lancet 1960: 2:325-329. 50. Gotto AM, Levy RI, John K, et al: On the protein defect in abetalipoproteinemia. N Engl J Med 1971; 284:813-818. 51. Schaefer EJ, Levy RI: Pathogenesis and management oflipoprotein disorders. N EnglJ Med 1985; 312:1300-1310. 52. Gouras P, Carr RE, Gunkel RD: Retinitis pigmentosa in abetalipoproteinemia: Effects of vitamin A. Invest Ophthalmol Vis Sci 1971; 10:784-793. 53. Carr RE: Abetalipoproteinemia and the eye, in Bergsma D, Bron AJ, Cotlier E (eds): T1he Eye and Inborn Errors ofMetabolism. Birth Defects: Original Article, Series XII, Vol 3. New York, Alan R Liss, Inc, 1976, pp 385-399. 54. Muller DPR, Lloyd JK, Bird AC: Long-term management of abetalipoproteinemia: Possible role of vitamin E. Arch Dis Child 1977; 52:209. 55. Bieri JG, Hoeg JM, Schaefer EJ, et al: Vitamin A and vitamin E replacement in abetalipoproteinemia. Ann Intern Med 1984; 100:238-239. 56. Bishara S, Merin S, Cooper M, et al: Combined vitamin A and E therapy prevents retinal electrophysiological deterioration in abetalipoproteinemia. Br J Ophthalmol 1982; 66:767-770. 57. Schaefer EJ: Diagnosis and management of ocular abnormalities in abetalipoproteinemia, in Zrenner E, Krastel H, Goebel HH (eds): Research in Retinitis Pigmen- tosa, Advances in Biosciences, Vol 62. Oxford, NY, Pergamon Press, 1987, pp 579-581. 58. Von Sallmann L, Gelderman AH, Laster L: Ocular histopathologic changes in a case of a-beta-lipoproteinemia (Bassen-Kornzweig syndrome). Doc Ophthalmol 1969; 26:451- 460. 59. Cogan DG, Rodrigues M, Chu FC, et al: Ocular abnormalities in abetalipoproteinemia: A clinicopathologic correlation. Ophthalmology 1984; 91:991-998. Rll) LLithl RhILodp))'iL(n('1U' I)CJ'C 387

60. Refsum S: Heredopathia atactica polyneuritiformis: A familial syndrome not hitherto described. Acta Psychiatr Scand (Suppl) 1946; 38:1-303. 61. Heredopathia atactica polyneuritiformis phytanic-acid storage disease, Ref- sum's disease: A biochemically well-defined disease with specific dietary treatment. Arch Neurol 1981; 38:605-606. 62. Klenk E, Kahlke W: Uber das Vorkommen der 3,7,11,15-tetramethylhexadecansaure (Phytansaure) in den Cholesterinestern und anderen Lipoidfraktionen der Organe bei einem Krakheitsfall unbekannter Genese Verdacht auf Heredopathia atactica polyneuritiformis (Refsum Syndrome). Hoppe Seylers Z Physiol Chem 1963; 333:133-139. 63. Eldjarn L, Stokke 0, Try K: Biochemical aspects ofRefsum's disease and principles for the dietary treatment, in Vinken PJ, Bruyn GW (eds): Handbook of Clinical Neurol- ogy. Chap 27, Amsterdam, North Holland Publishing Co, 1976, pp 519-541. 64. Toussant D, Davis P: An ocular pathologic study ofRefsum's syndrome. Aml Ophthal- mol 1971; 72:342-347. 65. Levy IS: Refsum's syndrome. Trans Ophthalmol Soc UK 1970; 90:181-186. 66. Donis-Keller H, Green P, Helms C, et al: A genetic linkage map of the human genome. CeU 1987; 51:319-337. 67. Sparkes RS, Mohandas T, Newman SL, et al: Assignment of the rhodopsin gene to human chromosome 3. Invest Ophthalmol Vis Sci 1986; 27:1170-1172. 68. Nathans J, Piantanida RL, Eddy RL, et al: Molecular genetics ofinherited variation in human color vision. Science 1986; 232:203-232. 69. Mandelbaum J: Dark adaptation: Some physiologic.and clinical considerations. Arch Ophthalmol 1941; 26:203-239. 70. Sloan LL: Light sense in pigmentary degeneration of the retina. Arch Ophthalmol 1956; 2:253-269. 71. Nathans SJ, Davenport CM, Maumenee IH, et al: Molecular genetics of human blue cone monochromacy. Science 1989; 245:831-838. 72. Reichel E, Bruce A, Sandberg MA, et al: An electroretinographic and molecular genetic study of X-linked cone degeneration. Am J Ophthalmol 1989; 108:540-547. 73. Applebury ML, Hargrave PA: Molecular biology of the visual pigments. Vision Res 1986; 26:1881-1895. 74. Nathans J, Hogness DS: Isolation and nucleotide sequence of the gene encoding human rhodopsin. Proc Natl Acad Sci USA 1984; 81:48514855. 75. Ferris FL, KassoffA, Bresnick GH, et al: New visual acuity charts for clinical research. Am J Ophthalmol 1982; 94:91-96. 76. Berson EL, Sandberg MA, Rosner B, et al: Natural course ofretinitis pigmentosa over a three year interval. Am J Ophthalmol 1985; 99:240-251. 77. Minkowski JS, Palise M, Guyton DL: Potential acuity meter using a minute aerial pinhole aperture. Ophthalmology 1983; 90:1360-1368. 78. Guyton D: Prediction of postoperative vision in cataract patients. Ophthalmol Clin North Am 1989; 2:431-440. 79. Rosner B: Fundamentals of Biostatistics, 3rd edition. Boston, Duxbury Press, 1989. 80. Cox DR: Analysis of Binary Data. London, Methuen, 1970. 81. Peto R, Pike M, Armitage P, et al: Design and analysis of randomized clinical trials requiring prolonged observation of each patient. Br J Cancer 1977; 35:1-39. 82. Massof RW, Finkelstein D: Two forms of autosomal dominant primary retinitis pigmentosa. Doc Ophthalmol 1981; 51:289-346. 83. Lyness AL, Ernst W, Quinlan MP, et al: A clinical, psychophysical, and electroretinographic survey of patients with autosomal dominant retinitis pigmentosa. Br J Ophthalmol 1985; 69:326-339. 84. Farber MD, Fishman GA, Weiss RW: Autosomal dominantly inherited retinitis pigmentosa: Visual acuity loss by subtype. Arch Ophthalmol 1985; 103:524-528. 85. Berson EL, Gouras P, Hoff M: Temporal aspects of electroretinogram. Arch Ophthal- mol 1969; 81:207-214. 388 Berson

86. Berson EL, Kanters L: Cone and rod responses in a family with recessively inherited retinitis pigmentosa. Arch Ophthalmol 1970; 84:288-297. 87. Young RW: Visual cell renewal systems and the problem of retinitis pigmentosa. Adv Exp Med Biol 1977; 77:93-113. 88. Bok D: Retinal photoreceptors-pigment epithelium interactions. Invest Ophthalmol Vis Sci 1985; 26:1659-1694. 89. Besharse JC: Photosensitive membrane turnover: Differentiated membrane domains and cell-cell interaction, in Adler R, Farber D (eds): The Retina: A Model for Cell Biological Studies, Part I. New York, Academic Press, 1986, pp 297-352. 90. Hargrave PA: Molecular dynamics of the rod cell, in Adler R, Farber D (eds): The Retina: A Model for Cell Biological Studies, Part I. New York, Academic Press, 1986, Dn 207-237. 91. Besharse JC, Horst CJ: The photoreceptor connecting cilium: A model for the transi- tion zone, in Bloodgood RA (ed): Ciliary and Flagellar Membranes. New York, Plenum Press, 1990, pp 389-417. 92. Hargrave PA, McDowell JH, Feldmann RJ, et al: Rhodopsin's protein and carbohy- drate structure: Selected aspects. Vision Res 1984; 24:1487-1499. 93. Fliesler SJ, Basinger SF: Tunicamycin blocks the incorporation of opsin into rod outer segment membranes. Proc Natl Acad Sci USA 1985; 82:1116-1120. 94. Fliesler SJ, Rayborn ME, Hollyfield JG: Membrane morphogenesis in retinal rod outer segments: Inhibition by tunicamycin. J CeU Biol 1985; 100:574-587. 95. Pratz EA, Miljanich GP, Ferris PP, et al: The structure of rhodopsin and its disposition in the rod outer segment disk membrane. Photochem Photobiol 1979; 29:661-670. 96. Clark SP, Molday RS: Orientation of membrane glycoproteins in sealed rod outer segment disks. Biochemistry 1979; 18:5868-5873. 97. Sandberg MA, Baruzzi C, Hanson AH, et al: Abnormal rod ERG diurnal rhythm in dominant retinitis pigmentosa. Invest Ophthalmol Vis Sci 1988; 29:494-498. 98. Scangos G, Bieberich C: Gene transfer into mice. Adv Genet 1987; 24:285-322. 99. Rosenfeld MG, Crenshaw EB, Lira SA, et al: Transgenic mice: Applications to the study of the nervous system. Ann Rev Neurosci 1988; 11:353-372. 100. Hogan B, Constantini F, Lacy E: Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory, 1986, pp 81-83. 101. Ferretti L, Karnik SS, Khorana HG, et al: Total synthesis of a gene for bovine rhodopsin. Proc Natl Acad Sci USA 1986; 83:599-603. 102. Oprian DD, Molday RS, Kaufman RJ, et al: Expression of a synthetic bovine rhodopsin gene in monkey kidney cells. Proc Natl Acad Sci USA 1987; 84:8874-8878. 103. Khorana HG, Knox BE, Nasi E, et al: Expression of a bovine rhodopsin in Xenopus oocytes: Demonstration of light dependent ionic currents. Proc Natl Acad Sci USA 1988; 85:7917-7921. 104. Berson EL: Light deprivation for early retinitis pigmentosa. Arch Ophthalmol 1971; 85:521-529. 105. Light deprivation and retinitis pigmentosa. Vision Res 1980; 20:1179-1184. 106. LaVail MM, Gorrin GM, Repaci MA, et al: Genetic regulation of light damage to photoreceptors. Invest Ophthalmol Vis Sci 1987; 28:1043-1048.