AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA WITH RHODOPSIN, VALINE-345-METHIONINE* BY Eliot L. Berson, MD, Michael A. Sandberg, PhD (BY INVITATION), AND (BY INVITATION) Thaddeus P. Dryja, MD INTRODUCTION
OUR
GROUP AND OTHERS HAVE DISCOVERED POINT MUTATIONS IN THE
rhodopsin gene in some patients with autosomal dominant retinitis pigmentosa.''-1 Each of these mutations is inherited together with the disease in the pedigrees so far examined. None of these mutations has been observed in unaffected individuals. These results support the idea that these mutations are the cause of some forms of autosomal dominant retinitis pigmentosa. Because it is now possible to classify some dominant cases according to their underlying genetic defect, it is appropriate to evaluate the clinical features exhibited by patients who have the same mutation. In this report we describe for the first time the ocular findings in a family with autosomal dominant retinitis pigmentosa caused by a point mutation in codon 345 of the rhodopsin gene. This mutation corresponds to a substitution of methionine for valine in the 345th amino acid of the rhodopsin protein. METHODS
Members of family 5067 (Fig 1) resided in the state of Maine and reported English ancestry. Each member who volunteered to participate completed a questionnaire asking age of onset of night blindness and age of *From the Berman-Gund Laboratory for the Study of Retinal Degenerations and the Howe Laboratory of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston. This research was supported in part by grants EY00169, EY02014, and EY08683 from the National Eye Institute, Bethesda, MD, and grants from the National Retinitis Pigmentosa Foundation, Baltimore and the George Gund Foundation, Cleveland. Dr Berson is a Research to Prevent Blindness Senior Scientific Investigator and Dr Dryja is a Research to Prevent Blindness-Dolly Green Scholar. TR. AM. OPHTH. Soc. vol. LXXXIX, 1991
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Pedigree of family 5067 with autosomal retinitis pigmentosa with valine-to-methionine change in amino acid 345 of rhodopsin. Solid symbols indicate affected individuals; open symbols indicate unaffected; oblique arrow indicates propositus, II-4.
onset of difficulty with side or peripheral vision. Members were evaluated with respect to best corrected Snellen visual acuities, Ferris visual acuities, and kinetic visual fields with a V4e white test light in the Goldmann perimeter. Following dilation and dark adaptation for 45 minutes, darkadapted psychophysical thresholds were measured in the GoldmannWeekers dark adaptometer to an 110 white test light fixated centrally or 70 above the fovea. Full-field electroretinograms (ERGs) were then elicited to 0.5-Hz flashes of blue light (1.2 log foot-lamberts X < 470 nm), 0.5-Hz flashes of white light (3.8 log foot-lamberts), and 30-Hz flashes of white light (3.8 log foot-lamberts) without computer averaging for responses : 10 ,N and with computer averaging for responses < 10 ,uV, as described previously."1 After electroretinographic testing, retinal acuity was obtained for each eye on a Snellen equivalent number chart with the
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Guyton-Minkowski potential acuity meter. Slit lamp examination was performed to determine the presence or absence of a central posterior subcapsular cataract in each eye. Ophthalmoscopic examination was then performed; each fundus was evaluated to determine whether cystoid macular edema could be seen and whether intraretinal bone spicule pigmentation was present in all four quadrants around the periphery. Because clinically affected members were found in three consecutive generations with father-to-son transmission in family 5067, it was clear that the disease was inherited by an autosomal dominant mode of transmission. Of interest was the history that the paternal grandparents of the propositus denied any history of retinitis pigmentosa, raising the possibility of a spontaneous mutation in the father of the propositus. These paternal grandparents were deceased, and deoxyribonucleic acid (DNA) was not available for analysis. The mother of the propositus denied any symptoms referable to retinitis pigmentosa. There was no family history of consanguinity. Leukocyte DNA from the propositus and his relatives, as well as from 149 other patients from separate families with autosomal dominant retinitis pigmentosa, were screened for mutations of the rhodopsin gene using the technique called single-strand conformation polymorphism (SSCP).12 DNA samples that had variant bands by SSCP were investigated further by directly sequencing the respective exon or exons, using methods previously reported.2 The same methods were used to evaluate DNA samples from 106 unrelated normal subjects who served as controls. RESULTS
All clinically affected individuals in family 5067 heterozygously carried a guanine-to-adenine (G-to-A) transition in the first nucleotide of codon 345. This nucleotide change would alter the specificity of the codon from valine to methionine (Fig 2). We have designated this mutation as rhodopsin, Val345Met. This point mutation was not present in two clinically normal siblings of the propositus nor in the mother of the propositus. In addition, this mutation was not observed in 149 other patients from separate families with autosomal dominant retinitis pigmentosa from across the United States nor in 106 subjects without retinitis pigmentosa who served as controls. This family also did not show any previously described point mutations in the rhodopsin gene in codons 23, 58, and 347. Clinical findings on history taking and ocular examination of seven affected members of family 5067 are summarized in Tables I and II. Six of seven were symptomatic with night blindness by age 18, and some also
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reported loss of side visionrby that age. Patient II-1 had visual acuity in the right eye reduced to 20/200, presumably due in part to a cataract, since retinal acuity was 20/70; patient I-1 was surgically aphakic in both eyes and still retained 20/50 and 20/30 vision with correction at age 58. Patients 11-2, II-1, and I-1, all aged 33 or older, showed intraretinal pigment in all four quadrants, while 2 of the 4 younger patients did not have this finding. None of these patients showed cystoid macular edema on ophthalmoscopic examination. Representative fundus photographs TABLE I: FINDINGS ON HISTORY TAKING IN AFFECTED PATIENTS
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from four of these patients are shown in Fig 3. Patient I-1, the oldest affected member of this family, had more bone spicule pigment than the younger affected relatives. Final dark-adapted thresholds, visual fields, and full-field ERGs are reported in Table III. Six of seven patients could perform dark-adaptation testing, and all had elevated final dark-adapted rod thresholds; patients II-4, II-2, and I-1 also had elevated cone thresholds (ie, > 2.5 log units above final normal dark-adapted thresholds). Those aged 33 and younger showed substantially larger visual fields than those aged 35 and 58. Some variation in the severity of the disease was noted in this family, since patient II-2 at age 33 retained a larger visual field area than patient II-4 at age 26. All patients had reduced ERG amplitudes; the youngest patient had the largest responses, and the oldest had the smallest responses. Again, some intrafamilial variability was observed, as patient II-2 at age 33 had a larger ERG than patient II-4 at age 26.
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Representative ERGs without computer averaging are illustrated in Fig 4; all patients had nondetectable rod-isolated responses to blue light and substantially reduced mixed cone and rod responses to 0.5-Hz flashes of white light. Cone-isolated responses to 30-Hz white flicker were normal in amplitude only in the youngest patient (III-1), aged 7; however, her cone b-wave implicit time was delayed, as was observed in the computeraveraged cone responses obtained from the other affected patients (Table III).
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Two siblings of the propositus had normal results on ocular examinations as well as normal final dark-adaptation rod thresholds and normal full-field ERGs. These two individuals did not carry this defect in the rhodopsin gene. DISCUSSION
The present study shows that all patients from a family with a G-to-A transition in codon 345 of the rhodopsin gene (corresponding to the substitution of methionine for valine in the 345th amino acid of rhodopsin or Val345Met) have ocular signs of retinitis pigmentosa. This mutation was not seen in two clinically unaffected siblings of the propositus, nor in 106 unrelated subjects without retinitis pigmentosa who served as controls. These results support the idea that this mutation is the cause of autosomal dominant retinitis pigmentosa in this family. Although loss of visual function was greatest in the two oldest members of this family and least in the youngest member of the family, some intrafamilial variability was noted. A 33-year-old patient (11-2) retained larger visual fields and larger ERG amplitudes than a 26-year-old sibling (II-4). This intrafamilial variability, in which an older patient shows less severe disease than a younger relative, is similar to that previously reported by us in families with other mutations of the rhodopsin gene, namely proline-23-histidine (ie, Pro23His) and proline-347-leucine (ie, Pro347Leu).3'4'7 This variability in severity suggests that factor(s) other than the gene defect itself may be involved in the clinical expression of this condition. Such factors could be other heritable traits that modify the rate of retinal degeneration, or they could be environmental variations (eg, exposure to sunlight, toxins) or variations in life-style (eg, diet, smoking). Among patients so far studied by us (Table IV), those with the Pro23His mutation have had larger 0.5-Hz ERG amplitudes, on average, at their initial visit than those with the Pro347Leu mutation, even though the former were, on average, 5 years older than the latter.7 ERGs recorded from patients from one family with rhodopsin, threonine-58-arginine (Thr58Arg), were also substantially larger, on average, than those recorded from the family with Val345Met in the present study or those recorded from a family with proline-347-serine (Pro347Ser). Since only a small number of patients have yet been reported with these mutations and since groups of patients with each mutation have not yet been followed prospectively in a longitudinal study, it remains to be established whether the severity of the disease or its rate of progression will correlate with a specific rhodopsin gene defect.
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TABLE IV: GROUP COMPARISONS OF ERGS OF PATIENTS WITH RHODOPSIN GENE MUTATIONS
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log amplitudes. The mechanism(s) by which these rhodopsin gene mutations lead to rod photoreceptor cell death remains to be defined. The rhodopsin protein normally traverses the rod outer segment membrane seven times (Fig 5) and is thought to be folded in three dimensions, with the first and seventh transmembrane segments in close proximity. Loops on the intradiscal side, as well as the region near the N-terminal tail, appear to be necessary for the proper folding of the moleculel3.'4; loops on the cytoplasmic side appear to interact with transducin as part of the phototransduction proCytoplasm CytoplasmHOOC-Ai)AO
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cess. 15 The proline-to-histidine mutation at position 23 may interfere with folding of rhodopsin, thereby modifying the capacity of the molecule to form a pocket to hold vitamin A. The change from a neutral threonine to a charged arginine may also alter the properties of the pocket. In the case of rhodopsin, Val345Met, as well as rhodopsin, Pro347Leu and rhodopsin, Pro347Ser, Hargrave and O'Brien'6 have speculated that the amino acids in the cytoplasmic domain near the carboxy terminus may affect proper transport of the rhodopsin molecule from the rough endoplasmic reticulum to the basal part of the rod outer segment. If this region of the rhodopsin molecule is defective, it is possible that the molecule is not released from the rough endoplasmic reticulum; the mutant opsin molecules may accumulate, possibly obstructing the synthesis and transport of normal opsin molecules to the outer segment. Studies of transgenic mice with mutant human rhodopsin gene constructs may help in defining pathogenesis. These rhodopsin gene mutations provide a new basis for classification of some forms of autosomal dominant retinitis pigmentosa. These diagnoses can be made through analysis of leukocyte DNA, thereby setting the stage for early diagnosis in families who wish to know whether or not their children are affected. Risk factor analyses of patients who have a more or less severe disease at a given age may help to reveal aggravating or ameliorating factors, with possible implications for therapy. SUMMARY
Rhodopsin gene mutations appear to cause some forms of autosomal dominant retinitis pigmentosa. In the family described, the mutation called rhodopsin, Val345Met segregated perfectly with the disease. All affected individuals had abnormal ERGs; the two oldest members of this family had more loss of function than the two youngest members. Some intrafamilial variability existed as an older member showed larger visual fields and ERG amplitudes than a younger member. This mutation was not seen in 106 control subjects nor in any other patients yet described with other rhodopsin gene mutations. Patients so far studied with rhodopsin, Val345Met, have smaller 0.5-Hz full-field ERG amplitudes, on average, than those with Pro23His or Thr58Arg and larger ERG amplitudes than those with Pro347Leu or Pro347Ser. These forms of retinitis pigmentosa can now be detected through analysis of leukocyte DNA.
128 1. 2.
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REFERENCES Dryja TP, McGee TL, Reichel E, et al: A point mutation of the rhodopsin gene in one form of retinitis pigmentosa. Nature 1990; 343:364-366. Dryja TP, McGee TL, Hahn LB, et al: Mutations within the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa. N Engl J Med 1990; 323:1302-1307. Berson EL: Ocular findings in a form of retinitis pigmentosa with a rhodopsin gene defect. Trans Am Ophthalmol Soc 1991; 88:355-388. Berson EL, Rosner B, Sandberg MA, et al: Ocular findings in patients with autosomal dominant retinitis pigmentosa and rhodopsin gene defect (Pro-23-His). Arch Ophthalmol 1991; 109:92-101. Heckenlively JR, Rodriguez JA, Daiger SP: Autosomal dominant sectoral retinitis pigmentosa: Two families with transversion mutation in codon 23 of rhodopsin. Arch Ophthalmol 1991; 109:84-91. Inglehearn CF, Bashir R, Lester DH, et al: A 3-bp deletion in the rhodopsin gene in a family with autosomal dominant retinitis pigmentosa. Am J Hum Genet 1991; 48:26-30. Berson EL, Rosner B, Sandberg MA, et al: Ocular findings in patients with autosomal dominant retinitis pigmentosa and rhodopsin, Proline-347-Leucine. Am J Ophthalmol 1991; 111:614-623. Dryja TP, Hahn LB, McGee TL, et al: Mutation spectrum of the rhodopsin gene among patients with autosomal dominant retinitis pigmentosa. Invest Ophthalmol Vis Sci (Suppl) 1991; 32:890. Bhattacharya SS, Inglehearn CF, Keen J, et al: Identification of novel rhodopsin mutations in patients with autosomal dominant retinitis pigmentosa. Invest Ophthalmol Vis Sci (Suppl) 1991; 32:890. Stone EM, Khadini P, Kimura AE, et al: A rapid denaturing gradient gel method of screening for rhodopsin gene mutations in patients with retinitis pigmentosa. Invest Ophthalmol Vis Sci (Suppl) 1991; 32:891. Berson EL, Sandberg MA, Rosner B, et al: Natural course of retinitis pigmentosa over a three-year interval. Am J Ophthalmol 1985; 99:240-251. Orita M, Suzuki Y, Sekiya T, et al: Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 1989; 5:874-879. Karnik SS, Sakmar TP, Chen HB, et al: Cysteine residues 110 and 187 are essential for the formation of correct structure in bovine rhodopsin. Proc Natl Acad Sci USA 1988; 85:8459-8463. Doi T, Molday RS, Khorana HG: Role of the intradiscal domain in rhodopsin assembly and function. Proc Natl Acad Sci USA 1990; 87:49914995. Franke RR, Konig B, Sakmar TP, et al: Rhodopsin mutants that bind but fail to activate transducin. Science 1990; 250:123-125. Hargrave PA, O'Brien PJ: Speculations on the molecular basis of retinal degeneration in retinitis pigmentosa, in RE Anderson, JG Hollyfield, MM LaVail (eds): Retinal Degenerations. Boca Raton, FL, CRC Press Inc., 1991, pp 517-528.
DISCUSSION DR JOHN R. HECKENLIVELY. Doctors Berson and co-workers should be commended for their strong efforts in utilizing molecular genetics to better characterize and understand the various forms of retinitis pigmentosa (RP). To place this work in perspective, the importance of this paper is that the molecular biologic techniques used to confirm the specific mutations in rhodopsin in these families with autosomal dominant RP represent the same type of technology that will shortly identify and give the pathophysiology of a large number of ophthalmologic diseases.
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McKusick's Catalog of Mendelian Inheritance in Man lists more than 200 retinal diseases with a hereditary component; fully 10% of the disorders listed in the catalog have an ophthalmologic component (Catalog of Mendelian Inheritance in Man. Baltimore, Johns Hopkins University Press, 1990). The gene sites for many of these diseases are currently being discovered at a rapid pace, and in the last year literally several new gene sites or genes have been reported monthly for ocular genetic disorders. Examples in the field of RP are the finding of Usher's syndrome type II with partial congenital deafness and RP on the long arm of chromosome 1 by Kimberling and associates (Genomics 1990; 7:245-249), at least two gene sites for X-linked RP reported by Musarella and colleagues (Genomics 1990; 8:286-296), and the isolation of the choroideremia gene on the long arm of the X chromosome by Cremers and associates (Nature 1990; 347:674-677) in the Netherlands. Mapping techniques have established that there are a minimum of three genetic types of autosomal dominant RP. The rhodopsin mutations on the long arm of chromosome 3; another form known to be near the rhodopsin gene on chromosome 3; and then, by exclusion, other types of dominant RP which do not map to chromosome 3. Preliminary studies suggest that approximately 20% of autosomal dominant RP have rhodopsin mutations; therefore, it would not be surprising if at least ten different genetic forms of autosomal dominant RP are found in the next 10 years. Molecular diagnosis is starting to unravel the diagnostic "black box" that we faced in trying to understand retinitis pigmentosa. Patients generally have had similar fundus findings, and we have had to use other traits such as inheritance pattern, electrophysiology, and psychophysics to further classify them. The following two cases of autosomal dominant RP would seem to be different according to the fundus findings, yet both of them have a rhodopsin mutation: Case 1 is a 28-year-old man with the codon-23 proline-to-histidine transversion with sectoral RP changes (Arch Ophthalmol 1991; 109:84-91). Case 2 is a 40-yearold woman with a codon-106 glycine-to-tryptophan transversion and pigmentary retinal degeneration in a pericentral distribution. As of last week, about 30 different rhodopsin mutations had been reported in different families with autosomal dominant RP. While these patients have rhodopsin mutations in common, each may be unique pathophysiologic mechanisms and it is possible that secondary factors are affecting the disease process. One of the powerful aspects of knowing the gene that is causing the RP is that other factors which are influencing gene expression, such as environmental factors and secondary gene expression, can be studied. Berson and colleagues in this study report marked differences in the expression of various rhodopsin mutations in their patients. Preliminary studies of rhodopsin mutations suggest that proline substitutions and loss of the molecule's disulfide bond are disruptive to its structure, and as reported by Bhattacharya at the Association for Research in Vision and Ophthalmology, a mutation at codon 296 results in a severe early-onset form of autosomal dominant RR
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The rhodopsin molecule is conserved across species and appears to be highly sensitive to base-pair changes which result in an alteration of the molecule's organization; an analogy has been made that it may be as susceptible to problems as the hemoglobin gene. The pathophysiologic mechanism that causes the RP in rhodopsin mutations is currently being studied, and inappropriate assembly or transport in the endoplasmic reticulum has been suggested. In summary, Berson and colleagues report the findings in a family with rhodopsin valine-to-methionine transition at codon 345 and compare the clinical findings in this family to those in other families with rhodopsin mutations that they have examined. This technology will undoubtedly lead to treatments, and possibly some cures, in the foreseeable future for a number of hereditary retinal diseases, and will allow us to better understand secondary factors, which also may be treatable, that influence the disease course. The investigators are to be congratulated for their fine contribution to the field of ophthalmology. DR ELIOT L. BERSON. Thank you very much Doctor Heckenlively for your kind remarks. All of us working on hereditary retinal diseases are enormously excited by the specificity offered by molecular biologic techniques in defining the biochemical bases of these diseases. The opportunity to diagnose some forms of autosomal dominant retinitis pigmentosa from analyses of leukocyte DNA opens a new chapter with respect to detection of affected patients at early stages. We can study patients who have minimal, if any, symptoms and search for risk factors that may correlate with a more or less severe clinical expression of the disease with possible implications for therapy. In the laboratory we have new opportunities as well. At the recent Association for Research in Vision and Ophthalmology meeting, we reported that we have injected a rhodopsin, proline-23-histidine human gene construct into the fertilized eggs of mice and produced a line of mice with retinal degeneration (Invest Ophthalmol Vis Sci [Suppl] 1991; 32:782). These transgenic mice can serve as a laboratory model that should aid us in our search for treatments for retinitis pigmentosa.
OUR
GROUP AND OTHERS HAVE DISCOVERED POINT MUTATIONS IN THE
rhodopsin gene in some patients with autosomal dominant retinitis pigmentosa.''-1 Each of these mutations is inherited together with the disease in the pedigrees so far examined. None of these mutations has been observed in unaffected individuals. These results support the idea that these mutations are the cause of some forms of autosomal dominant retinitis pigmentosa. Because it is now possible to classify some dominant cases according to their underlying genetic defect, it is appropriate to evaluate the clinical features exhibited by patients who have the same mutation. In this report we describe for the first time the ocular findings in a family with autosomal dominant retinitis pigmentosa caused by a point mutation in codon 345 of the rhodopsin gene. This mutation corresponds to a substitution of methionine for valine in the 345th amino acid of the rhodopsin protein. METHODS
Members of family 5067 (Fig 1) resided in the state of Maine and reported English ancestry. Each member who volunteered to participate completed a questionnaire asking age of onset of night blindness and age of *From the Berman-Gund Laboratory for the Study of Retinal Degenerations and the Howe Laboratory of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston. This research was supported in part by grants EY00169, EY02014, and EY08683 from the National Eye Institute, Bethesda, MD, and grants from the National Retinitis Pigmentosa Foundation, Baltimore and the George Gund Foundation, Cleveland. Dr Berson is a Research to Prevent Blindness Senior Scientific Investigator and Dr Dryja is a Research to Prevent Blindness-Dolly Green Scholar. TR. AM. OPHTH. Soc. vol. LXXXIX, 1991
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FIGURE 1
Pedigree of family 5067 with autosomal retinitis pigmentosa with valine-to-methionine change in amino acid 345 of rhodopsin. Solid symbols indicate affected individuals; open symbols indicate unaffected; oblique arrow indicates propositus, II-4.
onset of difficulty with side or peripheral vision. Members were evaluated with respect to best corrected Snellen visual acuities, Ferris visual acuities, and kinetic visual fields with a V4e white test light in the Goldmann perimeter. Following dilation and dark adaptation for 45 minutes, darkadapted psychophysical thresholds were measured in the GoldmannWeekers dark adaptometer to an 110 white test light fixated centrally or 70 above the fovea. Full-field electroretinograms (ERGs) were then elicited to 0.5-Hz flashes of blue light (1.2 log foot-lamberts X < 470 nm), 0.5-Hz flashes of white light (3.8 log foot-lamberts), and 30-Hz flashes of white light (3.8 log foot-lamberts) without computer averaging for responses : 10 ,N and with computer averaging for responses < 10 ,uV, as described previously."1 After electroretinographic testing, retinal acuity was obtained for each eye on a Snellen equivalent number chart with the
Retinitis Pigmentosa
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Guyton-Minkowski potential acuity meter. Slit lamp examination was performed to determine the presence or absence of a central posterior subcapsular cataract in each eye. Ophthalmoscopic examination was then performed; each fundus was evaluated to determine whether cystoid macular edema could be seen and whether intraretinal bone spicule pigmentation was present in all four quadrants around the periphery. Because clinically affected members were found in three consecutive generations with father-to-son transmission in family 5067, it was clear that the disease was inherited by an autosomal dominant mode of transmission. Of interest was the history that the paternal grandparents of the propositus denied any history of retinitis pigmentosa, raising the possibility of a spontaneous mutation in the father of the propositus. These paternal grandparents were deceased, and deoxyribonucleic acid (DNA) was not available for analysis. The mother of the propositus denied any symptoms referable to retinitis pigmentosa. There was no family history of consanguinity. Leukocyte DNA from the propositus and his relatives, as well as from 149 other patients from separate families with autosomal dominant retinitis pigmentosa, were screened for mutations of the rhodopsin gene using the technique called single-strand conformation polymorphism (SSCP).12 DNA samples that had variant bands by SSCP were investigated further by directly sequencing the respective exon or exons, using methods previously reported.2 The same methods were used to evaluate DNA samples from 106 unrelated normal subjects who served as controls. RESULTS
All clinically affected individuals in family 5067 heterozygously carried a guanine-to-adenine (G-to-A) transition in the first nucleotide of codon 345. This nucleotide change would alter the specificity of the codon from valine to methionine (Fig 2). We have designated this mutation as rhodopsin, Val345Met. This point mutation was not present in two clinically normal siblings of the propositus nor in the mother of the propositus. In addition, this mutation was not observed in 149 other patients from separate families with autosomal dominant retinitis pigmentosa from across the United States nor in 106 subjects without retinitis pigmentosa who served as controls. This family also did not show any previously described point mutations in the rhodopsin gene in codons 23, 58, and 347. Clinical findings on history taking and ocular examination of seven affected members of family 5067 are summarized in Tables I and II. Six of seven were symptomatic with night blindness by age 18, and some also
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reported loss of side visionrby that age. Patient II-1 had visual acuity in the right eye reduced to 20/200, presumably due in part to a cataract, since retinal acuity was 20/70; patient I-1 was surgically aphakic in both eyes and still retained 20/50 and 20/30 vision with correction at age 58. Patients 11-2, II-1, and I-1, all aged 33 or older, showed intraretinal pigment in all four quadrants, while 2 of the 4 younger patients did not have this finding. None of these patients showed cystoid macular edema on ophthalmoscopic examination. Representative fundus photographs TABLE I: FINDINGS ON HISTORY TAKING IN AFFECTED PATIENTS
PATIENT NQ PEDIGREE NO.
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7 III-1 1 19 II-8 2 26 II-6 3 26 II-4 4 33 5 II-2 35 IH-1 6 58 I-1 7 *Age at time of ocular examination. tNone designates patient has not yet
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from four of these patients are shown in Fig 3. Patient I-1, the oldest affected member of this family, had more bone spicule pigment than the younger affected relatives. Final dark-adapted thresholds, visual fields, and full-field ERGs are reported in Table III. Six of seven patients could perform dark-adaptation testing, and all had elevated final dark-adapted rod thresholds; patients II-4, II-2, and I-1 also had elevated cone thresholds (ie, > 2.5 log units above final normal dark-adapted thresholds). Those aged 33 and younger showed substantially larger visual fields than those aged 35 and 58. Some variation in the severity of the disease was noted in this family, since patient II-2 at age 33 retained a larger visual field area than patient II-4 at age 26. All patients had reduced ERG amplitudes; the youngest patient had the largest responses, and the oldest had the smallest responses. Again, some intrafamilial variability was observed, as patient II-2 at age 33 had a larger ERG than patient II-4 at age 26.
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Representative ERGs without computer averaging are illustrated in Fig 4; all patients had nondetectable rod-isolated responses to blue light and substantially reduced mixed cone and rod responses to 0.5-Hz flashes of white light. Cone-isolated responses to 30-Hz white flicker were normal in amplitude only in the youngest patient (III-1), aged 7; however, her cone b-wave implicit time was delayed, as was observed in the computeraveraged cone responses obtained from the other affected patients (Table III).
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Two siblings of the propositus had normal results on ocular examinations as well as normal final dark-adaptation rod thresholds and normal full-field ERGs. These two individuals did not carry this defect in the rhodopsin gene. DISCUSSION
The present study shows that all patients from a family with a G-to-A transition in codon 345 of the rhodopsin gene (corresponding to the substitution of methionine for valine in the 345th amino acid of rhodopsin or Val345Met) have ocular signs of retinitis pigmentosa. This mutation was not seen in two clinically unaffected siblings of the propositus, nor in 106 unrelated subjects without retinitis pigmentosa who served as controls. These results support the idea that this mutation is the cause of autosomal dominant retinitis pigmentosa in this family. Although loss of visual function was greatest in the two oldest members of this family and least in the youngest member of the family, some intrafamilial variability was noted. A 33-year-old patient (11-2) retained larger visual fields and larger ERG amplitudes than a 26-year-old sibling (II-4). This intrafamilial variability, in which an older patient shows less severe disease than a younger relative, is similar to that previously reported by us in families with other mutations of the rhodopsin gene, namely proline-23-histidine (ie, Pro23His) and proline-347-leucine (ie, Pro347Leu).3'4'7 This variability in severity suggests that factor(s) other than the gene defect itself may be involved in the clinical expression of this condition. Such factors could be other heritable traits that modify the rate of retinal degeneration, or they could be environmental variations (eg, exposure to sunlight, toxins) or variations in life-style (eg, diet, smoking). Among patients so far studied by us (Table IV), those with the Pro23His mutation have had larger 0.5-Hz ERG amplitudes, on average, at their initial visit than those with the Pro347Leu mutation, even though the former were, on average, 5 years older than the latter.7 ERGs recorded from patients from one family with rhodopsin, threonine-58-arginine (Thr58Arg), were also substantially larger, on average, than those recorded from the family with Val345Met in the present study or those recorded from a family with proline-347-serine (Pro347Ser). Since only a small number of patients have yet been reported with these mutations and since groups of patients with each mutation have not yet been followed prospectively in a longitudinal study, it remains to be established whether the severity of the disease or its rate of progression will correlate with a specific rhodopsin gene defect.
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TABLE IV: GROUP COMPARISONS OF ERGS OF PATIENTS WITH RHODOPSIN GENE MUTATIONS
NO. OF FAMILIES
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NO. OF CASES
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cess. 15 The proline-to-histidine mutation at position 23 may interfere with folding of rhodopsin, thereby modifying the capacity of the molecule to form a pocket to hold vitamin A. The change from a neutral threonine to a charged arginine may also alter the properties of the pocket. In the case of rhodopsin, Val345Met, as well as rhodopsin, Pro347Leu and rhodopsin, Pro347Ser, Hargrave and O'Brien'6 have speculated that the amino acids in the cytoplasmic domain near the carboxy terminus may affect proper transport of the rhodopsin molecule from the rough endoplasmic reticulum to the basal part of the rod outer segment. If this region of the rhodopsin molecule is defective, it is possible that the molecule is not released from the rough endoplasmic reticulum; the mutant opsin molecules may accumulate, possibly obstructing the synthesis and transport of normal opsin molecules to the outer segment. Studies of transgenic mice with mutant human rhodopsin gene constructs may help in defining pathogenesis. These rhodopsin gene mutations provide a new basis for classification of some forms of autosomal dominant retinitis pigmentosa. These diagnoses can be made through analysis of leukocyte DNA, thereby setting the stage for early diagnosis in families who wish to know whether or not their children are affected. Risk factor analyses of patients who have a more or less severe disease at a given age may help to reveal aggravating or ameliorating factors, with possible implications for therapy. SUMMARY
Rhodopsin gene mutations appear to cause some forms of autosomal dominant retinitis pigmentosa. In the family described, the mutation called rhodopsin, Val345Met segregated perfectly with the disease. All affected individuals had abnormal ERGs; the two oldest members of this family had more loss of function than the two youngest members. Some intrafamilial variability existed as an older member showed larger visual fields and ERG amplitudes than a younger member. This mutation was not seen in 106 control subjects nor in any other patients yet described with other rhodopsin gene mutations. Patients so far studied with rhodopsin, Val345Met, have smaller 0.5-Hz full-field ERG amplitudes, on average, than those with Pro23His or Thr58Arg and larger ERG amplitudes than those with Pro347Leu or Pro347Ser. These forms of retinitis pigmentosa can now be detected through analysis of leukocyte DNA.
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REFERENCES Dryja TP, McGee TL, Reichel E, et al: A point mutation of the rhodopsin gene in one form of retinitis pigmentosa. Nature 1990; 343:364-366. Dryja TP, McGee TL, Hahn LB, et al: Mutations within the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa. N Engl J Med 1990; 323:1302-1307. Berson EL: Ocular findings in a form of retinitis pigmentosa with a rhodopsin gene defect. Trans Am Ophthalmol Soc 1991; 88:355-388. Berson EL, Rosner B, Sandberg MA, et al: Ocular findings in patients with autosomal dominant retinitis pigmentosa and rhodopsin gene defect (Pro-23-His). Arch Ophthalmol 1991; 109:92-101. Heckenlively JR, Rodriguez JA, Daiger SP: Autosomal dominant sectoral retinitis pigmentosa: Two families with transversion mutation in codon 23 of rhodopsin. Arch Ophthalmol 1991; 109:84-91. Inglehearn CF, Bashir R, Lester DH, et al: A 3-bp deletion in the rhodopsin gene in a family with autosomal dominant retinitis pigmentosa. Am J Hum Genet 1991; 48:26-30. Berson EL, Rosner B, Sandberg MA, et al: Ocular findings in patients with autosomal dominant retinitis pigmentosa and rhodopsin, Proline-347-Leucine. Am J Ophthalmol 1991; 111:614-623. Dryja TP, Hahn LB, McGee TL, et al: Mutation spectrum of the rhodopsin gene among patients with autosomal dominant retinitis pigmentosa. Invest Ophthalmol Vis Sci (Suppl) 1991; 32:890. Bhattacharya SS, Inglehearn CF, Keen J, et al: Identification of novel rhodopsin mutations in patients with autosomal dominant retinitis pigmentosa. Invest Ophthalmol Vis Sci (Suppl) 1991; 32:890. Stone EM, Khadini P, Kimura AE, et al: A rapid denaturing gradient gel method of screening for rhodopsin gene mutations in patients with retinitis pigmentosa. Invest Ophthalmol Vis Sci (Suppl) 1991; 32:891. Berson EL, Sandberg MA, Rosner B, et al: Natural course of retinitis pigmentosa over a three-year interval. Am J Ophthalmol 1985; 99:240-251. Orita M, Suzuki Y, Sekiya T, et al: Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 1989; 5:874-879. Karnik SS, Sakmar TP, Chen HB, et al: Cysteine residues 110 and 187 are essential for the formation of correct structure in bovine rhodopsin. Proc Natl Acad Sci USA 1988; 85:8459-8463. Doi T, Molday RS, Khorana HG: Role of the intradiscal domain in rhodopsin assembly and function. Proc Natl Acad Sci USA 1990; 87:49914995. Franke RR, Konig B, Sakmar TP, et al: Rhodopsin mutants that bind but fail to activate transducin. Science 1990; 250:123-125. Hargrave PA, O'Brien PJ: Speculations on the molecular basis of retinal degeneration in retinitis pigmentosa, in RE Anderson, JG Hollyfield, MM LaVail (eds): Retinal Degenerations. Boca Raton, FL, CRC Press Inc., 1991, pp 517-528.
DISCUSSION DR JOHN R. HECKENLIVELY. Doctors Berson and co-workers should be commended for their strong efforts in utilizing molecular genetics to better characterize and understand the various forms of retinitis pigmentosa (RP). To place this work in perspective, the importance of this paper is that the molecular biologic techniques used to confirm the specific mutations in rhodopsin in these families with autosomal dominant RP represent the same type of technology that will shortly identify and give the pathophysiology of a large number of ophthalmologic diseases.
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McKusick's Catalog of Mendelian Inheritance in Man lists more than 200 retinal diseases with a hereditary component; fully 10% of the disorders listed in the catalog have an ophthalmologic component (Catalog of Mendelian Inheritance in Man. Baltimore, Johns Hopkins University Press, 1990). The gene sites for many of these diseases are currently being discovered at a rapid pace, and in the last year literally several new gene sites or genes have been reported monthly for ocular genetic disorders. Examples in the field of RP are the finding of Usher's syndrome type II with partial congenital deafness and RP on the long arm of chromosome 1 by Kimberling and associates (Genomics 1990; 7:245-249), at least two gene sites for X-linked RP reported by Musarella and colleagues (Genomics 1990; 8:286-296), and the isolation of the choroideremia gene on the long arm of the X chromosome by Cremers and associates (Nature 1990; 347:674-677) in the Netherlands. Mapping techniques have established that there are a minimum of three genetic types of autosomal dominant RP. The rhodopsin mutations on the long arm of chromosome 3; another form known to be near the rhodopsin gene on chromosome 3; and then, by exclusion, other types of dominant RP which do not map to chromosome 3. Preliminary studies suggest that approximately 20% of autosomal dominant RP have rhodopsin mutations; therefore, it would not be surprising if at least ten different genetic forms of autosomal dominant RP are found in the next 10 years. Molecular diagnosis is starting to unravel the diagnostic "black box" that we faced in trying to understand retinitis pigmentosa. Patients generally have had similar fundus findings, and we have had to use other traits such as inheritance pattern, electrophysiology, and psychophysics to further classify them. The following two cases of autosomal dominant RP would seem to be different according to the fundus findings, yet both of them have a rhodopsin mutation: Case 1 is a 28-year-old man with the codon-23 proline-to-histidine transversion with sectoral RP changes (Arch Ophthalmol 1991; 109:84-91). Case 2 is a 40-yearold woman with a codon-106 glycine-to-tryptophan transversion and pigmentary retinal degeneration in a pericentral distribution. As of last week, about 30 different rhodopsin mutations had been reported in different families with autosomal dominant RP. While these patients have rhodopsin mutations in common, each may be unique pathophysiologic mechanisms and it is possible that secondary factors are affecting the disease process. One of the powerful aspects of knowing the gene that is causing the RP is that other factors which are influencing gene expression, such as environmental factors and secondary gene expression, can be studied. Berson and colleagues in this study report marked differences in the expression of various rhodopsin mutations in their patients. Preliminary studies of rhodopsin mutations suggest that proline substitutions and loss of the molecule's disulfide bond are disruptive to its structure, and as reported by Bhattacharya at the Association for Research in Vision and Ophthalmology, a mutation at codon 296 results in a severe early-onset form of autosomal dominant RR
130
Berson & Dryja
The rhodopsin molecule is conserved across species and appears to be highly sensitive to base-pair changes which result in an alteration of the molecule's organization; an analogy has been made that it may be as susceptible to problems as the hemoglobin gene. The pathophysiologic mechanism that causes the RP in rhodopsin mutations is currently being studied, and inappropriate assembly or transport in the endoplasmic reticulum has been suggested. In summary, Berson and colleagues report the findings in a family with rhodopsin valine-to-methionine transition at codon 345 and compare the clinical findings in this family to those in other families with rhodopsin mutations that they have examined. This technology will undoubtedly lead to treatments, and possibly some cures, in the foreseeable future for a number of hereditary retinal diseases, and will allow us to better understand secondary factors, which also may be treatable, that influence the disease course. The investigators are to be congratulated for their fine contribution to the field of ophthalmology. DR ELIOT L. BERSON. Thank you very much Doctor Heckenlively for your kind remarks. All of us working on hereditary retinal diseases are enormously excited by the specificity offered by molecular biologic techniques in defining the biochemical bases of these diseases. The opportunity to diagnose some forms of autosomal dominant retinitis pigmentosa from analyses of leukocyte DNA opens a new chapter with respect to detection of affected patients at early stages. We can study patients who have minimal, if any, symptoms and search for risk factors that may correlate with a more or less severe clinical expression of the disease with possible implications for therapy. In the laboratory we have new opportunities as well. At the recent Association for Research in Vision and Ophthalmology meeting, we reported that we have injected a rhodopsin, proline-23-histidine human gene construct into the fertilized eggs of mice and produced a line of mice with retinal degeneration (Invest Ophthalmol Vis Sci [Suppl] 1991; 32:782). These transgenic mice can serve as a laboratory model that should aid us in our search for treatments for retinitis pigmentosa.
