SHORT COMMUNICATION Pro-347-Arg Mutation of the Rhodopsin Gene in Autosomal Dominant Retinitis Pigmentosa ANDREAS GAL, *,’ ANDREAS ARTLICH,* MICHAEL LuDwlc,j- GRUNTER NIEMEYER,~ KLAUS OLEK, 5 EBERHARD SCHWINGER, * AND ALBERT SCHINZEL~~ */nstitut
fur Humangenetik, Medizinische Universitat, Liibeck, Federal Republic of Germany; tlnstitut fiir experimentelle Hd;matologie and Slnstitut fslr Klinische Biochemie der Universit;Tt, Bonn, Federal Republic of Germany; §Augenklinik and Vnstitut fur Medizinische Genetik der Universitat, Zurich, Switzerland Received
April
15, 1991;
It has been shown recently that autosomal dominant retinitis pigmentosa may be caused by point mutations of the rhodopsin gene in a portion of families. In this communication, a large six-generation family with autosomal dominant RP is described. Molecular analysis by PCR amplification followed by restriction digestion or heteroduplex analysis suggested a point mutation in codon 347, in which two different mutations (Pro-347-Ser and Pro-347-Leu) have already been reported. Direct sequencing of the patients’ DNA revealed a previously undescribed CCG + CGG transversion in codon 347 predicting a Pro + Arg substitution. Ophthalmological data of the patients are summarized and compared to those of patients with other mutations in the rhodopsin gene. Q 1s~ Academic PAM, IUC.
Retinitis pigmentosa (RP), the most common hereditary degenerative disorder of the retina, is both clinically and genetically heterogeneous. It has been shown recently that autosomal dominant RP may be caused by point mutations in the gene for rhodopsin in about 20% of the cases in the United States (Dryja et al., 1990a). Until now, four different point mutations (at codons 23,58, and 347) have been identified in patients with autosomal dominant RP in the United States (Dryja et al., 1990a,b). Additionally, a 3-bp deletion at codon 255 has recently been reported in a British family with autosomal dominant RP (Inglehearn et al., 1991). Specific amplification of genomic DNA fragments by polymerase chain reaction (PCR) allows easy detection of mutations and largescale screening of patients. We analyzed about 90 unrelated patients with autosomal dominant RP from Western Europe for the presence of the five muta1To whom correspondence and reprint requests should be addressed at the Institut fiir Humangenetik, MUL, Ratzeburger Allee 160, W 2400 Liibeck 1, FRG. Fax: D-451-500-4187.
GENOMICS 11,468-470 (1991) o&3&7543/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
Inc. reserved.
revisedJune
12, 1991
tions given above (Farrar et al., 1990; Orth et ai, 1991, and unpublished). In a Swiss patient with autosomal dominant RP, a previously unreported point mutation at codon 347 was detected. This codon (CCG) specifies the amino acid proline at the carboxyl-terminal end of the rhodopsin polypeptide. Two different mutations in this codon (CCG-tTCG or +CTG), predicting a substitution of proline by serine (Pro347-Ser) and leucine (Pro-347-Leu), respectively, have already been reported (Dryja et al., 199Oa). We synthesized a pair of oligonucleotide primers (5’-AACTGCATGCTCACCACCA-3’ and 5’-TATGTGACTTCGTTCATTCTG-3’) to amplify a 219-bp fragment encompassing codon 347. Fortuitously, codon 347 in exon 5 of the rhodopsin gene (Nathans and Hogness, 1984) is part of an MspI recognition sequence (CCGG). Thus, digestion of the PCR product by MspI yields two fragments of 99 and 120 bp. Since both mutations at codon 347 given above abolish the MspI cutting site, patients with these nucleotide changes show a heterozygous pattern consisting of three fragments (Fig. 1A and Dryja et uZ., 1990a). Another means of detecting these point mutations relies on heteroduplex formation between nonhomologous DNA strands in PCR (Keen et al., 1991). Using this approach, the single base pair mismatch at codon 347 can be visualized on nondenaturing ethidium bromide-stained acrylamide gels (Fig. 1B). The mutation at codon 347 could be observed in the DNA of the Swiss patient by both methods (Figs. 1A and B). However, the electrophoretic mobility of the heteroduplexes differed slightly from that seen in the (more frequent) Pro-347-Leu ‘mutation (data not shown). Direct sequencing of the 219-bp fragment obtained after PCR amplification of the patient’s DNA revealed a previously undescribed C-to-G transversion of the second nucleotide of codon 347 (CCG + CGG; Fig. 1C). This mutation should result in a re-
SHORT
A
469
COMMUNICATION
‘A
B
c
G
T
14
-309
t
219~
C
-211
c G
-201 .I90 460
I
123456
C/G c
::::
-219 -120 l 99
C
GT
C G G
I
G T
G G A
460
1234
Pro-347
Pro347-Arg
FIG. 1. Detection of Pro-34’7-Arg mutation by enzyme cleavage (A), heteroduplex analysis (B), or direct sequencing (C) after PCR amplification. For details, see text. (A) Lane 1, control DNA, lanes 2-6, family members VI.1,2,3,5, and 7, respectively. (B) Lane 1, VI.l; lane 2, VI.3; lane 3, control DNA, lane 4, MW size marker. The sizes of DNA fragments/PCR products in A and B are given in bp. (C) Left, VI.3; right, VI.1. The DNA sequence found is indicated in the middle with a bracket for codon 347.
placement of the nonpolar proline by the polar amino acid arginine. The proband belongs to a large six-generation Swiss family segregating for autosomal dominant RP (Fig. 2) that was first described 45 years ago (Ammann, 1946). All six affected family members studied, but none of the ophthalmologically normal relatives of the patient, carry the Pro-347-Arg mutation as demonstrated by restriction enzyme cleavage or direct sequencing or both after PCR amplification of genomic DNA. We have not found this mutation in nearly 100 other Western European autosomal dominant RP patients (Orth et al., 1991, and unpublished data). Previous medical records on eight patients from generations III-V were available (Ammann, 1946, and unpublished), while six other patients from generations V-VI were examined ophthalmologically in 1990. All patients complained of night blindness from early age. Mild myopia and marked myopic oblique astigmatism were detected in most patients. Cataracta complicata was seen frequently above the age of
40, while mild irregularities of the posterior lens capsule could be detected at even younger age. Loss of visual field was reported from the second decade onward, resulting in an extreme constriction to tunnel vision at the age of 50-60. The Ganzfeld rod-cone electroretinogiaphy (Niemeyer, 1979) revealed gross retinal abnormality. The cone and particularly the rod ERG was greatly reduced from the early twenties on, and the cone b-waves were markedly reduced and the implicit time delayed at the age of 18-22 years, The age of virtual blindness was between 40 and 60. Interestingly, in this family, patients below the age of 40 do not show the retinal hyperpigmentation typical of RP (“sine pigment0 form”). Nevertheless, most of the patients present with typical atrophic changes and pigmentation at later age. Autosomal dominant RP shows a considerable intra- and interfamilial clinical variability. The demonstration of allelic heterogeneity renders studies possible on the correlation between genotype and disease phenotype. Until now, ophthalmological examinations have not allowed correlation of a specific RP phenotype to any of the known mutations. In most of the patients with a rhodopsin mutation, however, a diffuse and severe loss in the function of rods, the only retinal cells known to express rhodopsin, has been observed early in life (D type autosomal dominant RP). Two of 14 patients/families with the Pro-23-His et al, mutation showed sectoral RP (Heckenlively 1991).
FIG. 2. Pedigree of the family modified and completed). 1: patient
studied (after studied.
Ammann,
1946,
A comparison of the published medical records reveals that patients with the Pro-23-His mutation or codon 255 deletion have on average a less severe disease than those with any of the three known mutations at codon 347. At present, due to the limited amount of information, it is difficult to make any comparison of the phenotypes observed in conjunction
470
SHORT
COMMUNICATION
with the three different mutations of codon 347. It is interesting, however, that patients with the Pro-347Arg mutation have only minimal retinal hyperpigmentation, at least in their second and third decades, in contrast to the heavy deposition of pigment seen in patients with the Pro-347-Leu mutation (E. L. Berson, personal communication). The effect of the Pro-347-Arg change on the secondary structure of rhodopsin can be manifold. Nonpolar amino acids, like proline, tend to cluster together on the inside of molecule. Certainly, the shape of the carboxyl-terminal end of the rhodopsin polypeptide may be critically altered by the presence of the basic arginine, thereby leading to a considerable decrease in the stability of the protein. From a functional point of view, it is known that the C-terminal region, close to codon 347, contains specific sites that are subject to light-dependent phosphorylation by rhodopsin kinase. Second, a catalytic site that promotes GTP-GDP exchange by transducin has also been located to the cytoplasmic domain of rhodopsin. The clustering of mutations at codon 347 is interesting and can suggest that this part of the gene is especially prone to molecular rearrangements. Indirect support for this assumption comes from the observation that in patients with the Pro-347-Leu mutation no linkage disequilibrium has been observed between the mutation and an intragenic rhodopsin polymorphism (Dryja et al., 199Oa), making it likely that this mutation arose independently more than one time.
REFERENCES 1.
AMMANN, E. (1946). Zur Vererbung der Hemeralopia hereditaria und tapeto-retinalen Degeneration. Ophthulmologica (Basel) 112: 78-81.
2.
DRYJA, T. P., MCGEE, T. L., HAHN, L. B., COWLEY, G. S., OLSSON, J. E., REICHEL, E., SANDBERG, M. A., AND BERSON, E. L. (1990a). Mutations within the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa. N. Engl. J. Med. 323: 1302-1307. DRYJA, T. P., MCGEE, T. L., REICHEL, E., HAHN, L. B., CowLEY, G. S., YANDELL, D. W., SANDBERG, M. A., AND BERSON, E. L. (199Ob). A point mutation of the rhodopsin gene in one form of retinitis pigmentosa. Nature 343: 364-366.
3.
4.
5.
6.
7.
Genet. 7: 5. 8.
9.
ACKNOWLEDGMENTS 10. This study was financially supported by the Deutsche Forschungsgemeinschaft (Ga 210/5-l). Special thanks are due to the members of the family studied and to Mrs. Christina Fasser (Schweizerische RP-Vereinigung) for their cooperation.
FARRAR, G. J., KENNA, P., REDMOND, R., MCWILLZAM, P., BRADLEY, D. G., HUMPHRIES, M. M., SHARP, E. M., ING~HEARN, C. F., BASHIR, R., JAY, M., WATI~~, A., LUDWIG, M., SCHINZJXL, A., SAMANNS, CH., GAL, A., BHATTACHARYA, S. S., AND HUMPHFUES, P. (1990). Autosomal dominant retinitis pigmentosa: Absence of the rhodopsin proline + histidine substitution (codon 23) in pedigrees from Europe. Am. J. Hum. Genet. 47: 941-945. HECKENLIVELY, J. R., RODRIQUEZ, J. A., AND DAIGER, S. P. (1991). Autosomal dominant sectoral retinitis pigmentosa: Two families with transversion mutation in codon 23 of rhodopsin. Arch. Ophthalmol. 109: 84-91. INGLEHEARN, C. F., BASH& R., LESTER, D. H., JAY, M., BIRD, A. C., AND BHATTACHARYA, S. S. (1991). A 3-bp deletion in the rhodopsin gene in a family with autosomal dominant retinitis pigmentosa. Am. J. Hum. Genet. 48: 26-30. KEEN, J., LESTER, D., INGIXHEARN, C., CURTIS, A., AND BHA’ITACHARYA, S. S. (1991). Rapid detection of single base mismatches as heteroduplexes on Hydrolink gels. Trends NATHANS, J., AND HOGNESS, D. S. (1984). Isolation and nucleotide sequence of the gene encoding human rhodopsin. Proc. Natl. Acad. Sci. USA 81: 4851-4855. N~MEXER, G. (1979). Information von der Netzhaut durch Elektroretinographie. A. v. Graefe’s Arch. Klin. Exp. Ophtti. 211: 129-137. ORTH, U., SAMANNS, CH., GUSSECK, H., NEWER, G., LUDWIG, M., MEITINGER, T., SCHINZXL, A., SCHWINGER, E., AND GAL, A. (1991). Autosomal-dominant erbliche Retinopathia pigmentosa ist genetisch heterogen. Fortsch. Ophthalmol., in press.
fur Humangenetik, Medizinische Universitat, Liibeck, Federal Republic of Germany; tlnstitut fiir experimentelle Hd;matologie and Slnstitut fslr Klinische Biochemie der Universit;Tt, Bonn, Federal Republic of Germany; §Augenklinik and Vnstitut fur Medizinische Genetik der Universitat, Zurich, Switzerland Received
April
15, 1991;
It has been shown recently that autosomal dominant retinitis pigmentosa may be caused by point mutations of the rhodopsin gene in a portion of families. In this communication, a large six-generation family with autosomal dominant RP is described. Molecular analysis by PCR amplification followed by restriction digestion or heteroduplex analysis suggested a point mutation in codon 347, in which two different mutations (Pro-347-Ser and Pro-347-Leu) have already been reported. Direct sequencing of the patients’ DNA revealed a previously undescribed CCG + CGG transversion in codon 347 predicting a Pro + Arg substitution. Ophthalmological data of the patients are summarized and compared to those of patients with other mutations in the rhodopsin gene. Q 1s~ Academic PAM, IUC.
Retinitis pigmentosa (RP), the most common hereditary degenerative disorder of the retina, is both clinically and genetically heterogeneous. It has been shown recently that autosomal dominant RP may be caused by point mutations in the gene for rhodopsin in about 20% of the cases in the United States (Dryja et al., 1990a). Until now, four different point mutations (at codons 23,58, and 347) have been identified in patients with autosomal dominant RP in the United States (Dryja et al., 1990a,b). Additionally, a 3-bp deletion at codon 255 has recently been reported in a British family with autosomal dominant RP (Inglehearn et al., 1991). Specific amplification of genomic DNA fragments by polymerase chain reaction (PCR) allows easy detection of mutations and largescale screening of patients. We analyzed about 90 unrelated patients with autosomal dominant RP from Western Europe for the presence of the five muta1To whom correspondence and reprint requests should be addressed at the Institut fiir Humangenetik, MUL, Ratzeburger Allee 160, W 2400 Liibeck 1, FRG. Fax: D-451-500-4187.
GENOMICS 11,468-470 (1991) o&3&7543/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
Inc. reserved.
revisedJune
12, 1991
tions given above (Farrar et al., 1990; Orth et ai, 1991, and unpublished). In a Swiss patient with autosomal dominant RP, a previously unreported point mutation at codon 347 was detected. This codon (CCG) specifies the amino acid proline at the carboxyl-terminal end of the rhodopsin polypeptide. Two different mutations in this codon (CCG-tTCG or +CTG), predicting a substitution of proline by serine (Pro347-Ser) and leucine (Pro-347-Leu), respectively, have already been reported (Dryja et al., 199Oa). We synthesized a pair of oligonucleotide primers (5’-AACTGCATGCTCACCACCA-3’ and 5’-TATGTGACTTCGTTCATTCTG-3’) to amplify a 219-bp fragment encompassing codon 347. Fortuitously, codon 347 in exon 5 of the rhodopsin gene (Nathans and Hogness, 1984) is part of an MspI recognition sequence (CCGG). Thus, digestion of the PCR product by MspI yields two fragments of 99 and 120 bp. Since both mutations at codon 347 given above abolish the MspI cutting site, patients with these nucleotide changes show a heterozygous pattern consisting of three fragments (Fig. 1A and Dryja et uZ., 1990a). Another means of detecting these point mutations relies on heteroduplex formation between nonhomologous DNA strands in PCR (Keen et al., 1991). Using this approach, the single base pair mismatch at codon 347 can be visualized on nondenaturing ethidium bromide-stained acrylamide gels (Fig. 1B). The mutation at codon 347 could be observed in the DNA of the Swiss patient by both methods (Figs. 1A and B). However, the electrophoretic mobility of the heteroduplexes differed slightly from that seen in the (more frequent) Pro-347-Leu ‘mutation (data not shown). Direct sequencing of the 219-bp fragment obtained after PCR amplification of the patient’s DNA revealed a previously undescribed C-to-G transversion of the second nucleotide of codon 347 (CCG + CGG; Fig. 1C). This mutation should result in a re-
SHORT
A
469
COMMUNICATION
‘A
B
c
G
T
14
-309
t
219~
C
-211
c G
-201 .I90 460
I
123456
C/G c
::::
-219 -120 l 99
C
GT
C G G
I
G T
G G A
460
1234
Pro-347
Pro347-Arg
FIG. 1. Detection of Pro-34’7-Arg mutation by enzyme cleavage (A), heteroduplex analysis (B), or direct sequencing (C) after PCR amplification. For details, see text. (A) Lane 1, control DNA, lanes 2-6, family members VI.1,2,3,5, and 7, respectively. (B) Lane 1, VI.l; lane 2, VI.3; lane 3, control DNA, lane 4, MW size marker. The sizes of DNA fragments/PCR products in A and B are given in bp. (C) Left, VI.3; right, VI.1. The DNA sequence found is indicated in the middle with a bracket for codon 347.
placement of the nonpolar proline by the polar amino acid arginine. The proband belongs to a large six-generation Swiss family segregating for autosomal dominant RP (Fig. 2) that was first described 45 years ago (Ammann, 1946). All six affected family members studied, but none of the ophthalmologically normal relatives of the patient, carry the Pro-347-Arg mutation as demonstrated by restriction enzyme cleavage or direct sequencing or both after PCR amplification of genomic DNA. We have not found this mutation in nearly 100 other Western European autosomal dominant RP patients (Orth et al., 1991, and unpublished data). Previous medical records on eight patients from generations III-V were available (Ammann, 1946, and unpublished), while six other patients from generations V-VI were examined ophthalmologically in 1990. All patients complained of night blindness from early age. Mild myopia and marked myopic oblique astigmatism were detected in most patients. Cataracta complicata was seen frequently above the age of
40, while mild irregularities of the posterior lens capsule could be detected at even younger age. Loss of visual field was reported from the second decade onward, resulting in an extreme constriction to tunnel vision at the age of 50-60. The Ganzfeld rod-cone electroretinogiaphy (Niemeyer, 1979) revealed gross retinal abnormality. The cone and particularly the rod ERG was greatly reduced from the early twenties on, and the cone b-waves were markedly reduced and the implicit time delayed at the age of 18-22 years, The age of virtual blindness was between 40 and 60. Interestingly, in this family, patients below the age of 40 do not show the retinal hyperpigmentation typical of RP (“sine pigment0 form”). Nevertheless, most of the patients present with typical atrophic changes and pigmentation at later age. Autosomal dominant RP shows a considerable intra- and interfamilial clinical variability. The demonstration of allelic heterogeneity renders studies possible on the correlation between genotype and disease phenotype. Until now, ophthalmological examinations have not allowed correlation of a specific RP phenotype to any of the known mutations. In most of the patients with a rhodopsin mutation, however, a diffuse and severe loss in the function of rods, the only retinal cells known to express rhodopsin, has been observed early in life (D type autosomal dominant RP). Two of 14 patients/families with the Pro-23-His et al, mutation showed sectoral RP (Heckenlively 1991).
FIG. 2. Pedigree of the family modified and completed). 1: patient
studied (after studied.
Ammann,
1946,
A comparison of the published medical records reveals that patients with the Pro-23-His mutation or codon 255 deletion have on average a less severe disease than those with any of the three known mutations at codon 347. At present, due to the limited amount of information, it is difficult to make any comparison of the phenotypes observed in conjunction
470
SHORT
COMMUNICATION
with the three different mutations of codon 347. It is interesting, however, that patients with the Pro-347Arg mutation have only minimal retinal hyperpigmentation, at least in their second and third decades, in contrast to the heavy deposition of pigment seen in patients with the Pro-347-Leu mutation (E. L. Berson, personal communication). The effect of the Pro-347-Arg change on the secondary structure of rhodopsin can be manifold. Nonpolar amino acids, like proline, tend to cluster together on the inside of molecule. Certainly, the shape of the carboxyl-terminal end of the rhodopsin polypeptide may be critically altered by the presence of the basic arginine, thereby leading to a considerable decrease in the stability of the protein. From a functional point of view, it is known that the C-terminal region, close to codon 347, contains specific sites that are subject to light-dependent phosphorylation by rhodopsin kinase. Second, a catalytic site that promotes GTP-GDP exchange by transducin has also been located to the cytoplasmic domain of rhodopsin. The clustering of mutations at codon 347 is interesting and can suggest that this part of the gene is especially prone to molecular rearrangements. Indirect support for this assumption comes from the observation that in patients with the Pro-347-Leu mutation no linkage disequilibrium has been observed between the mutation and an intragenic rhodopsin polymorphism (Dryja et al., 199Oa), making it likely that this mutation arose independently more than one time.
REFERENCES 1.
AMMANN, E. (1946). Zur Vererbung der Hemeralopia hereditaria und tapeto-retinalen Degeneration. Ophthulmologica (Basel) 112: 78-81.
2.
DRYJA, T. P., MCGEE, T. L., HAHN, L. B., COWLEY, G. S., OLSSON, J. E., REICHEL, E., SANDBERG, M. A., AND BERSON, E. L. (1990a). Mutations within the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa. N. Engl. J. Med. 323: 1302-1307. DRYJA, T. P., MCGEE, T. L., REICHEL, E., HAHN, L. B., CowLEY, G. S., YANDELL, D. W., SANDBERG, M. A., AND BERSON, E. L. (199Ob). A point mutation of the rhodopsin gene in one form of retinitis pigmentosa. Nature 343: 364-366.
3.
4.
5.
6.
7.
Genet. 7: 5. 8.
9.
ACKNOWLEDGMENTS 10. This study was financially supported by the Deutsche Forschungsgemeinschaft (Ga 210/5-l). Special thanks are due to the members of the family studied and to Mrs. Christina Fasser (Schweizerische RP-Vereinigung) for their cooperation.
FARRAR, G. J., KENNA, P., REDMOND, R., MCWILLZAM, P., BRADLEY, D. G., HUMPHRIES, M. M., SHARP, E. M., ING~HEARN, C. F., BASHIR, R., JAY, M., WATI~~, A., LUDWIG, M., SCHINZJXL, A., SAMANNS, CH., GAL, A., BHATTACHARYA, S. S., AND HUMPHFUES, P. (1990). Autosomal dominant retinitis pigmentosa: Absence of the rhodopsin proline + histidine substitution (codon 23) in pedigrees from Europe. Am. J. Hum. Genet. 47: 941-945. HECKENLIVELY, J. R., RODRIQUEZ, J. A., AND DAIGER, S. P. (1991). Autosomal dominant sectoral retinitis pigmentosa: Two families with transversion mutation in codon 23 of rhodopsin. Arch. Ophthalmol. 109: 84-91. INGLEHEARN, C. F., BASH& R., LESTER, D. H., JAY, M., BIRD, A. C., AND BHATTACHARYA, S. S. (1991). A 3-bp deletion in the rhodopsin gene in a family with autosomal dominant retinitis pigmentosa. Am. J. Hum. Genet. 48: 26-30. KEEN, J., LESTER, D., INGIXHEARN, C., CURTIS, A., AND BHA’ITACHARYA, S. S. (1991). Rapid detection of single base mismatches as heteroduplexes on Hydrolink gels. Trends NATHANS, J., AND HOGNESS, D. S. (1984). Isolation and nucleotide sequence of the gene encoding human rhodopsin. Proc. Natl. Acad. Sci. USA 81: 4851-4855. N~MEXER, G. (1979). Information von der Netzhaut durch Elektroretinographie. A. v. Graefe’s Arch. Klin. Exp. Ophtti. 211: 129-137. ORTH, U., SAMANNS, CH., GUSSECK, H., NEWER, G., LUDWIG, M., MEITINGER, T., SCHINZXL, A., SCHWINGER, E., AND GAL, A. (1991). Autosomal-dominant erbliche Retinopathia pigmentosa ist genetisch heterogen. Fortsch. Ophthalmol., in press.
