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Autosomal Dominant Retinitis Pigmentosa: Four New Mutations in Rhodopsin, One of Them in the Retinal Attachment Site T. J. KEEN, C. F. INGLEHEARN, D. H. LESTER,’ R. BASHIR, M. JAY,* A. C. BIRD,* 8. JAY, AND S. S. BHATTACHARYA Molecular Genetics Unit, Department of Human Genetics, 19 Claremont Place, Newcastle upon Tyne, United Kingdom; *Department of Clinical Ophthalmology, Moorfields Eye Hospital, City Road, London, EC1 V 2PD, United Kingdom Received

March

INTRODUCTION

Retinitis pigmentosa (RP) is a subgroup of the retinal photoreceptor dystrophies that give rise to loss of night vision early in the course of the disease, followed by loss of the peripheral visual field and characteristic changes in the ocular fundus. There is great variation in the age of onset of symptoms and severity of functional deficit. The condition affects about 15000 of the population of the United Kingdom, and accounts for just over 2% of registered blind (Department of Health and Social Security, 1988). RP may be subdivided according to the mode of inheritance and there is good evidence of heterogeneity within each address: Scotland.

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category. In the autosomal dominant class, which accounts for around 20% of primary RP (Jay, 1982), families are further classified into D-type and R-type categories (Lyness et al, 1985) or into the approximately corresponding type I and type II categories respectively (Massof and Finkelstein, 1981). In Dtype ADRP, night blindness is consistently recognized in the first decade, with diffuse and severe loss of rod function but relatively good preservation of cone function. The R-type disease is characterized by patchy and equal loss of both rod and cone function, with a wide variation in age of onset. McWilliam and co-workers (1989) demonstrated linkage between ADRP and the chromosome 3q marker Cl7 (D3S47) in a large Irish D-type ADRP family (TCDMl). Since then, other ADRP families have shown linkage (with D3S47) at zero recombination, linkage with recombination, and no linkage, demonstrating an underlying genetic heterogeneity. The three Cl7-linked families with no recombination are D type (McWilliam et al., 1989; Lester et al., 1990; Inglehearn et al., 1991), while the Cl7-linked family with recombination is classified as type II (Olsson et al., 1990). Four unlinked families have also been classified R type or type II (Inglehearn et al., 1990; Blanton et a& 1990; Farrar et al., 1990; Lester et al., 1990). Other reported families remain unclassified by these criteria. More recently, Dryja et al. (1990a) reported a mutation in codon 23 of the rhodopsin gene in 12% of a panel of 148 ADRP patients. Rhodopsin maps close to locus D3S47 on chromosome 3q (Nathans et aZ., 1986) and was therefore a good candidate gene in C17linked families. Subsequently, a further mutation in codon 347 was reported in the same panel, accounting for 6% of patients, while three other mutations have been reported in families or individual patients (Dryja et al., 199Ob; Inglehearn et al., 1991). However, haplotype data suggest that individuals with the codon 23

Several mutations in the rhodopsin gene in patients affected by autosomal dominant retinitis pigmentosa (ADRP) have recently been described. We report four new rhodopsin mutations in ADRP families, initially identified as heteroduplexed PCR fragments on hydrolink gels, One is an in-frame 12-bp deletion of codons 68 to 71. The other three are point mutations involving codons 190, 211, and 296. Each alters the amino acid encoded. The codon 190 mutation has been detected in 2 from a panel of 34 ADRP families, while the remaining mutations were seen in single families. This suggests that, consistent with a dominant condition, no single mutation will account for a large fraction of ADRP cases. The base substitution in codon 296 alters the lysine residue that functions as the attachment site for ll&-retinal, mutating it to glutamic acid. This mutation occurs in a family with an unusually severe phenotype, resulting in early onset of disease and cataracts in the third or fourth decade of life. This result demonstrates a correlation between the location of the mutation and the severity of phenotype in rhodopsin RP. Q lssi ~eademic POW, I~C.

1 Present Edinburgh,

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osss-7543/91$3.00 by Academic Press, Inc. in any form reserved.

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TABLE Oligonucleotide A: B: c: D: E: F: G: H: I: J:

T C A C T T T A G T

T A C C T C C C A T

C T T A G C A G G C

G G C G G A C C T G

C T T G C G G C A T

A T C G T A G A G T

G T C A G C C G C C

C C C G T C T C T A

A T A G T A C G T T

1

Primer T G G G C T T T G T

mutation shared common ancestors, as did those with the codon 347 mutation (Dryja et al., 199Ob). Using a method developed in this laboratory (Keen et al., 1991), we have screened the rhodopsin gene for mutations in a panel of 34 unrelated ADRP families.

T C A A C G G G T C

Sequences C T C A C G A G C T

T G C T A C G C C G

T A C G A T G A T C

G T C T G C G T T A

: c

G

T

FIG. 1. (a) Hydrolink gel showing the bandshift in the PCR product of primers E and F (315 bp) in individuals with the exon 3 codon 190 mutation. Lanes 1 and 3 are affected heterozygotes, while lane 2 is a normal homozygote. (b) Sequence from ADRP25 exon 3, generated from primer using a PCR of primers E and I (646 bp) as a template, and showing the codon 190 mutation showing the codon 190 mutation on the noncodmg strand (i.e., CTG to

TTG).

G G C A C T C C G A

T T C A C C C T C G

G G A G C C A A A G

G C T C T A G C G C

C

C

A

T

Exonic regions from affected patients are amplified by polymerase chain reaction (PCR) and then separated on nondenaturing hydrolink gels which resolve from the main band heteroduplexed fragments of the normal with the mutated strand. Using this method, we have detected four new mutations in exons 1, 3, and 4, which are almost certainly the cause of ADRP in these families. PATIENTS

b

G C T G T C T T G C

AND

METHODS

Genomic DNAs were prepared from peripheral blood obtained from patients on a panel of 34 ADRP families. The families in which mutations were found are as follows. ADRPlO has been classified as D type, with night blindness apparent from early childhood. Onset of the disease is severe with patients often registered blind by the fourth decade. Cataracts are frequently reported in the third and fourth decades. ADRP39 is a small pedigree with a mild phenotype, with relatively good preservation of visual field in the fourth decade of life. Families ADRPlO and 39 are English, ADRP25 and ADRP30 are Sardinian, and ADRP38 is Scottish. With the exception of ADRPlO, these families are, as yet, phenotypically unclassified. Mutations were detected using the previously described method of DNA heteroduplex detection on ethidium bromide-stained hydrolink gels (Keen et al., 1991). Pairs of oligonucleotide primers were synthesized surrounding exonic regions to give PCR products in the range of 200-300 bp (Table 1). PCR was carried out using a compromise two-stage reaction profile for all reactions, consisting of 30 cycles of 94°C for 30 s followed by 60°C for 5 min. We have found this to be adequate for products up to 600 bp using 20-bp primers of 5060% GC content. Heteroduplex molecules form after the final denaturing step in the PCR. When a final incubation at 68°C for 1 h was included, this reduced the amount of ghost banding due to secondary structure in the DNA molecule. Aliquots of the PCR reaction mixes were then electrophoresed on D-5000 hydrolink gels (AT Biochem) cast in 24-cm-long l-mm-thick Hoefer vertical gel tanks. Typically, gels were run for 1600-2000 Vh. In

RHODOPSIN

FIG. 2. ADRP25 pedigree to the numbered individuals individuals.

superimposed above them.

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on an agarose gel, showing TaqI-digested exon 3 PCRs from primers E and F. Tracks correspond A band representing the incut PCR product can be seen segregating in heterozygous affected

our experience, in comparison with polyacrylamide, hydrolink D-5000 gels made according to manufacturers’ instructions appear to resolve less widely by size in the range 200-400 bp, yet resolve more substantially differences in DNA conformation and secondary structure. The limited degree of size resolution exhibited by the hydrolink gels compared to that of polyacrylamide allowed a degree of standardization of running conditions independent of the absolute size of the PCR product, while still resulting in identification of heterozygous individuals. Once detected, mutations were sequenced by double-strand sequencing of heat-denatured PCR fragments using an end-labeled primer internal to those used for amplification, as described previously (Lester et al., 1990). Sequence was generated with sequenase (T7 DNA polymerase) according to the manufacturer’s instructions. Mutations were sequenced on both strands to confirm their location. RESULTS

Mutations were detected in exon 3 of three separate families. Two of the families (ADRP25 and ADRP30) showed the same mutation, and as both were from Sardinia, it would seem likely that they shared a common ancestor. The mutation in these families produces a slight but clearly recognizable band shift on the gel (Fig. la). Sequencing revealed a point mutation changing codon 190 from GAC to AAC, resulting in the substitution of an asparagine residue for the normal aspartate (Fig. lb). Fortuitously, the mutated nucleotide lies within a Z’aqI restriction site. Hence the mutation in affected individuals can be detected by the presence of an extra band when TaqI-digested

exon 3 PCR product is run on a 3% Nuseive/l% agarose gel (Fig. 2). The second exon 3 mutation was detected in affected members of a British ADRP family (ADRP38). In this case the appearance of the band shift in affected individuals is quite distinct compared to that of the codon 190 mutation. A second band of almost the same intensity is seen above the main band (Fig. 3a). Sequencing revealed a point mutation, changing codon 211 from CAC (histidine) to CCC (proline) as shown in Fig. 3b. In exon 1 a deletion was detected in one affected individual from an ADRP family (designated ADRP39). The band shift pattern on the hydrolink gel was clearly characteristic of a deletion rather than of a point mutation. There was slight separation of the normal and mutated homoduplex strands, while the two heteroduplex bands, normal plus with deleted minus and deleted plus with normal minus, were greatly retarded in mobility (Fig. 4a). Sequencing with an internal primer in exon 1 indicated that 12 bp were deleted, removing codons 68-71, which code for Leu-Arg-Thr-Pro (Fig. 4b). Sequencing from the other side of the deletion was carried out to confirm its boundaries. A 3-bp deletion has previously been reported by this laboratory, at codon 255/256 (Inglehearn et al., 1991). We have additionally located a point mutation in the 3’ part of this exon in a British family (ADRPlO). The heteroduplexed PCR product in this case was a separate discrete band migrating slower than the main band (Fig. 5a). Sequencing showed a point mutation changing codon 296 from AAG to GAG corresponding to an amino acid change of lysine to glutamic acid (Fig. 5b).

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ADRPlO. The opsin messenger RNA is translated in the rough endoplasmic reticulum (ER), where it isprocessed to form a three-dimensional structure that allows binding of II-&-retinal, giving rise to a functionally active rhodopsin molecule. This is then trans-

a

1

2

A C

FIG. 3. (a) Hydrolink gel showing the bandshift in the PCR product of primers E and F in individuals with the exon 3 codon 211 mutation (ADRP38). Lanes 1, 2, and 3 are amplified from affected individuals, while lane 4 shows a normal PCR product. (h) Sequence from exon 3 in family ADRP38, generated from primer E using PCR-amplified DNA from primers D and I (696 bp) as a template. The codon 211 mutation is shown in the coding strand (i.e., CAC-CCC).

In each case, with the exception of ADRP39, where only one affected member was available, the mutations found segregated in only affected members of the pedigree and not in any of the normal siblings. It is thus highly likely that these are the causative mutations of ADRP in these families. The locations of these mutations in the rhodopsin molecule are shown in Fig. 6. DISCUSSION This study presents four new rhodopsin mutations that appear to be the cause of ADRP in 5 of the 34 families studied. In each case the mutations alter a part of the molecule for which a function has been previously postulated. This is particularly true of the codon 296 lysine to glutamic acid mutation in

A C

c

A

FIG. 4. (a) Hydrolink gel resolving the band shift in the PCR product of primers B and C (295 bp) in the individual from family ADRP39 with a deletion of codons 68-71 in exon 1. Lane 1 is a normal PCR product, while lane 2 has the deletion. (b) Sequence of the exon 1 deletion on the coding strand, generated with primer B from a template PCR of primers A and C (477 bp). The correct sequence is written at the left-hand side, with, from the point of the deletion start, the mutated sequence alongside it. These two sequences are seen superimposed on the gel.

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FIG. 6. (a) The ADRPlO pedigree is shown superimposed on a hydrolink gel with PCR-amplified product from primers H and I (204 bp) of ADRPlO individuals. The band shift, with the heteroduplex running above the homoduplex band, is seen to segregate with ADRP. (b) Sequence from exon 4 in an affected individual from ADRPlO, generated from primer I using a template PCR-amplified using primers G and J (1382 bp). The codon 296 mutation is shown on the noncoding strand (i.e., TTC-CTC).

ported through the Golgi complex into vesicles that carry it to the base of the connecting cilium, where it fuses with the plasma membrane (St. Jules et aZ., 1989; Hargrave and O’Brien, 1991). Lysine 296 functions as the site of attachment for the retinal molecule to the opsin polypeptide (see Fig. 6). This residue is conserved not only in all vertebrate and invertebrate opsins but even in bacteriorhodopsin, the photosynthetic pigment of Halobacteria (Ovchinnikov, 1982). Glutamic acid lacks the amino group to which retinal would normally bind. Further, substitution of Lys296 with glycine and alanine in in vitro studies gave molecules unable to generate a light-sensitive chromophore (Zhukovsky et aZ., 1991). Thus, the codon 296 mutation observed must certainly render this attachment impossible. Without a source of retinal, newly synthesized opsin has been shown to accumulate in the rough ER of the isolated rat retina (St. Jules et al., 1986). The accumulation of the defective opsin, a molecule that accounts for 80% of the protein of the rod outer segment (Basinger et al., 1976), must surely interfere with normal cellular processes in the rod. The mutation at codon 190, found in families ADRP25 and 30, is located in the second intradiscal domain (see Fig. 6). In in vitro studies, deletion of rhodopsin residues 189 and 190 gave a protein that

failed to generate the chromophore when incubated with retinal (Doi et al., 1990). In the same study substitution of alanine at Asp190 gave a molecule that generated the chromophore to only lo-20% of wildtype levels. Glycosylation patterns of both were different from those of wild-type protein and consistent with those expected when a protein does not migrate from the ER to the Golgi body. The deletion of codons 68-71 in ADRP39 is in the cytoplasmic loop connecting the first and second transmembrane helices (see Fig. 6). This is the most conserved region on the cytoplasmic surface and has been suggested to be a point of interaction with cytoplasmic proteins (Applebury and Hargrave, 1986). However, it seems likely that the removal of these amino acids has an effect on protein folding in addition to any functional significance this region may have in the signal transduction pathway. Likewise, the mutation in ADRP38 at codon 211 in the fifth transmembrane domain (Fig. 6) is likely to have an effect on protein folding. Invariant prolines are found in five of the transmembrane helices (Applebury and Hargrave, 1986). Proline residues are thought to introduce 20” bends into the molecule and it has been suggested that kinked helices are important for the formation of the pocket where the retinal group will lie (Dratz and Hargrave, 1983). The codon 211 histi-

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\

Retinal attachment site LYS

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INTRADISCAL FIG. 6. Diagrammatic residues are evolutionarily

representation conserved.

of the rhodopsin

molecule,

dine to proline substitution introduces an extra proline residue within three amino acids of one of the invariant prolines, and is therefore likely to have a substantial effect on structure. In conclusion, it seems reasonable to speculate that each of the mutations described would result in a protein with structural abnormalites that would compromise binding of 11-&-retinal and would therefore not be exported to the rod discs with the same efficiency as wild-type rhodopsin. Exactly how this leads to rod cell death is unknown at this time. However, it is interesting to note that rhodopsin RP patients with an amino acid substitution at the retinal attachment site itself have a phenotype more severe than that of other rhodopsin RP patients. It seems likely that some rhodopsin mutations give a protein that functions and is exported to the rod outer segment at reduced efficiency, while this mutation probably renders export from the rough ER impossible (Hargrave and O’Brien, 1991). It has also been suggested that failure to regenerate rhodopsin following bleaching may give rise to photoreceptor disc instability, as is seen in experimental vitamin A deficiency (Darling and Gibbons, 1961). Three of the seven ADRP families reported by this laboratory as having rhodopsin mutations have been classified D type, while the others are, as yet, unclassified (Inglehearn et al., 1990, Keen et al., 1991). Therefore, the D/R clinical classification may corre-

DOMAIN with

known

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spond to the underlying rhodopsin/nonrhodopsin genetic heterogeneity. The fact that only a proportion of ADRP families have rhodopsin mutations (as demonstrated by linkage), and that in most cases each of these has a different mutation, makes routine ADRP diagnosis at the DNA level difficult. However, the method used in this study provides a relatively rapid screen, not only for known rhodopsin mutations but also for previously undiscovered ones. It distinguishes between point mutations and deletion/insertion events, and localizes each to within a small region of coding sequence. By this technique, or using conventional linkage analysis with chromosome 3q probes, it is now possible to offer informed counseling and a molecular diagnosis for some ADRP families. With the identification of rhodopsin as one ADRP causing gene, other proteins involved in the phototransduction pathway are now candidates for the remaining forms of RP.

ACKNOWLEDGMENTS We are grateful to the Wellcome Foundation, National Betinitis Pigmentosa Foundation Fighting Blindness USA, and the George Gund Foundation for funding this research. We thank Dr. Jeremy Nathans for making available to us unpublished sequence data. Our thanks also to Helen A&ford and Brenda Lauffart for technical assistance. We are indebted to Pauline Battista and Bachelle Townsley for typing this manuscript.

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REFERENCES 1. APPLEBURY, M. L., AND HAFUXAVE, P. A. (1986). Molecular biology of the visual pigments. Vision Res. 26: 1881-1895. 2. BASINGER, S., BOK, D., AND HALL, H. (1976). Rhodopsin in the rod outer segment plasma membrane. J. Cell Bial. 69: 29-42.

3. BLANTON, S. H., COTTINGHAM, A. W., GIESENSCHLAG, N., HECKENLIVELY, J. R., HUMPHRIES, P., AND DAIGER, S. P. (1990). Further evidence of exclusion of linkage between type II autosomal dominant retinitis pigmentosa (ADRP) and D3S47 on 3q. Genomics 8: 179-181. 4. BOTERMANS, C. H. G. (1972). Primary pigmentary retinal degeneration and its association with neurological diseases. In “Handbook of Clinical Neurology 13” (P. J. Vinken and G. W. Bruyn, Eds.), pp. 148-379, Elsevier, Amsterdam/New York. 5. BUNDEY, S., AND CREWS, S. J. (1984). A study of retinitis pigmentosa in the City of Birmingham. II. Clinical and genetic heterogeneity. J. Med. Genet. 21: 421-428. 6. DARLING, J. E., AND GIBBONS, I. R. (1961). The effect of vitamin A deficiency on the fine structure of the retina. In “Structure of the Eye” (G. Smelser, Ed.), pp. 85-89, Academic Press, New York. 7. Department of Health and Social Security (1988). Causes of blindness and partial sight among adults in 1976/77 and 1980/81 England. Her Majesty’s Stationary Office; Table 5. 8. DOI, T., MOLDAY, R. S., AND KHORANA, H. G. (1990). Role of the intradiscal domain in rhodopsin assembly and function. Proc. Natl. Acad. Sci. USA 87: 4991-4995. 9. DFZATZ,E. A., AND HARGIUVE, P. A. (1983). The structure of rhodopsin and the rod outer segment disc membrane. Trends Biochem. Sci. 8: 128-131. 10. DRYJA, T. P., MCGEE, T. L., REICHEL, E., HAHN, L. B., CowLEY, G. S., YANDELL, D. W., SANDBERG, M. A., AND BERSON, E. L. (1990a). A point mutation of the rhodopsin gene in one form of retinitis pigmentosa. Nature 343: 364-366. 11. DRYJA, T. P., MCGEE, T. L., HAHN, L. B., COWLEY, G. S., OLLSEN, J. E., REICHEL, E., SANDBERG, M. A., AND BERSON, E. L. (1990b). Mutations within the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa. N. Engl. J. Med. 323: 1302-1307. 12. FARRAR, G. J., MCWILLIAM, P., BRADLEY, D. G., KENNA, P., LAWLER, M., SHARP, E. M., HUMPHRIES, M. M., EIBERG, H., CONNEALLY, P. M., TROFA~ER, J. A., AND HUMPHRIES, P. (1990). Autosomal dominant retinitis pigmentosa: Linkage to rhodopsin and evidence for genetic heterogeneity. Genamics 8:35-40. 13. HARGRAVE, P. A., AND O’BRIEN, P. J. (1991). Speculations on the molecular basis of retinal degeneration in retinitis pigmentosa. In “Retinal Degenerations” (R. E. Anderson, J. G. Hollyfield, and M. M. LaVail, Eds.), CRC Press, Boca Raton, FL, in press. 14. INGLEHEARN, C. F., JAY, M., LESTER, D. H., BASHIR, R., JAY, B., BIRD, A. C., WRIGHT, A. F., EVANS, H. J., PAPIHA, S. S., AND BHATTACHARYA, S. S. (1990). No evidence for linkage between late onset autosomal dominant retinitis pigmentosa

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and Chromosome 3 locus D3S47 (C17). Evidence for genetic heterogeneity. Genamics 6: 168-173. INGLEHEARN, C. F., BASHIR, R., LESTER, D. H., JAY, M., BIRD, A. C., AND BHA~ACHARYA, S. S. (1991). A three basepair deletion in the rhodopsin gene in a family with autosoma1 dominant retinitis pigmentosa. Am. J. Hum. Genet. 48: 26-30. JAY, M. (1982). Figures and fantasies: The frequencies of the different genetic forms of retinitis pigmentosa. Birth Dejects Orig. Artic. Series 18: 167-173. JIMINEZ, J. B., SAMANNS, C., WAN, A., PONGRATZ, J., OLSSON, J. E., DICKINSON, P., BU~RY, R., GAL, A., AND DENTON, M. J. (1991). No evidence of linkage between the locus for autosomal dominant retinitis pigmentosa and D3S47 (C17) in three Australian families. Hum. Genet. 86: 265-267. KEEN, J., LESTER, D. H., INGLEHEARN, C. F., CURTIS, A., AND BHATTACHARYA, S. S. (1991). Rapid detection of single base mismatches as heteroduplexes on hydrolink gels. Trends Genet.

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Autosomal dominant retinitis pigmentosa: four new mutations in rhodopsin, one of them in the retinal attachment site.

Several mutations in the rhodopsin gene in patients affected by autosomal dominant retinitis pigmentosa (ADRP) have recently been described. We report...
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