© 1992 Oxford University Press
Human Molecular Genetics, Vol. 1, No. 1 41—45
A completed screen for mutations of the rhodopsin gene in a panel of patients with autosomal dominant retinitis pigmentosa Chris F.lngleheam, T.Jeffrey Keen, Rumaisa Bashir, Marcelle Jay2, Fred Fitzke2, Alan C.Bird2, Alex Crombie1 and Shomi Bhattacharya* Department of Human Genetics, department of Ophthalmology, University of Newcastle upon Tyne and department of Clinical Ophthalmology, Moorfields Eye Hospital, London, UK Received November 15, 1991; Revised and Accepted December 16, 1991
ABSTRACT
INTRODUCTION Retinitis pigmentosa (RP) is a term used to denote a number of inherited disorders causing progressive loss of peripheral vision and night-blindness, in many cases leading to blindness in later life. Classically they are characterised by bone-spicule like pigmentation observed in the mid-periphery of the retina in later stages of the disease process. Other clinical features include narrowed retinal vessels and a severely altered or non-recordable rod electro-retinogram (ERG)1. RP can be inherited in X-linked, dominant, and recessive forms, and is also found in syndromes with other phenotypic defects2. The autosomal dominant class (adRP) accounts for around 22% of RP patients3. Attempts have been made to sub-divide this category further on the basis of gene penetrance4, age of onset5'6 and distribution of pigment in the affected retina7. Two such classification systems are currently in common usage with approximately corresponding categories. D-type ('diffuse') or * To whom correspondence should be addressed
type I adRP causes diffuse and severe loss of rod function, but cone function is retained until much later in the disease process. Patients consistently experience night-blindness within the first ten years of life, though pigmentary changes may not become evident until the third decade. In the D-type, ERG and psychophysical testing shows that rod function is abnormal over the whole fundus with relative preservation of cone function7. In R-type ('regional') or type 2 adRP the age of onset varies both between and within families. Indeed, some R-type families include individuals who appear asymptomatic yet have transmitted the disease to the next generation. The R-type disease causes 'regional' or patchy and equal loss of rod and cone function. Dtype disease is estimated to account for 22% of adRP 78 , with the bulk of the remainder being R type disease. Another rarer category of adRP is sectorial RP, which consistantly affects only one or two quadrants of the retina within a family, with the remaining retina left intact. It is evident that sectorial RP as described is also heterogeneous. Some authors have reserved the term for disease in which there is no progression into the spared segments and the ERG has a normal implicit time9. Others have used the term to denote non-progressive disease but with dysfunction in the unaffected regions10'1'. The term has also been used to include disease in which one part of the retina is affected earlier and more profoundly than the remainder, but which is progressive and eventually affects the whole visual field12. Linkage analysis in families with adRP has demonstrated that some have a genetic defect located on chromosome 3q13'14 while others do not". Soon after the report of a genetic linkage, a base substitution was identified in codon 23 of the rhodopsin gene, which maps to chromosome 3q, in 17 of 148 apparendy unrelated adRP patients in the USA16. Rhodopsin is the rod photopigment which initiates the visual transduction cascade. It is composed of seven helical transmembrane domains, bounded by cytoplasmic and intradiscal loops (see figure 5), and represents 80—90% of the constituent protein of the rod outer segment17. The same US laboratory has since reported a further three point mutations in adRP, two in codon 347 and one in codon 5818. One of the codon 347 mutations and the codon 58 mutation occurred in single families, while the other codon 347 mutation was found in 8 out of 150 adRP patients. However, haplotyping of probes from
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Recently it has been demonstrated that some families with autosomal dominant retinitis pigmentosa (adRP) have mutations in the rhodopsin gene while others do not. Previously we have identified six such mutations in seven adRP families in this laboratory, one of which was previously described in US patients. We now present a completed screen of the rhodopsin gene in a panel of 39 adRP families, by a rapid screening technique which will be of use for routine diagnosis. Nine different mutations were ultimately found, in a total of twelve of the 39 families. These include the six previously identified mutations, in codons 6 8 - 7 1 , 190, 211, 255, 296 and 347, two new ones in codons 53 and 106, and another mutation first identified in a single US patient, in codon 58. Thus approximately 30% of adRP families have 'Rhodopsin RP' while the remainder probably have a defect elsewhere in the genome. Of those families in which rhodopsin mutations have been found, four have been classified D type, three as sectorial RP and the remainder are of uncertain classification. All families excluded from chromosome 3q by linkage have been classified R type. These data suggest a correlation between clinical sub-classification and the underlying rhodopsin/non-rhodopsin heterogeneity.
42 Human Molecular Genetics, Vol. 1, No. 1 around the rhodopsin gene region suggested that individuals with the codon 23 mutation may have a common ancestor, as was the case in patients with the commoner codon 347 mutation19. A screen of some 91 European adRP families revealed none with a codon 23 mutation20. We have described previously five new rhodopsin mutations and also observed the commoner codon 347 mutation in European adRP patients2122. These were observed in a panel of 39 adRP families for which genealogical evidence is available to demonstrate that they are not related in recent generations. We now present a screen of the entire coding sequence of rhodopsin in patients from every family on that panel. The results obtained give an estimate for the frequency of rhodopsin RP as a fraction of total adRP. In addition, the completed screen demonstrates an apparent correlation between phenotype and genotype in adRP, and provides an insight into the population dynamics of a dominant genetic condition.
Figure 1. A graphic representation of the genomic structure of rhodopsin, drawn approximately to scale. Hatched areas represent exons, which are numbered 1 to 5 reading from 5' to 3'. Oligomers used in this study are shown as lines above (coding strand) and below (non-coding strand) the graphic, and are assigned letters which correspond to those shown in table 1.
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RESULTS Of the families screened in this project, seven have already been reported to have mutations in rhodopsin. These consist of families with a codon 347 point mutation24, a deletion of codon 25521, point mutations in codons 190 (two families), 211 and 296, and a deletion of codons 68-71 2 2 . In completing the screen, we have identified mutations in a further five families. In adRP 28, a small clinically unclassified pedigree of Scottish origin, a bandshift was identified between primers A and K in exon 1. When sequenced, this was found to be due to a CCC to CGC (Pro to Arg) mutation in codon 53 (Figure 2). In family adRP 44, an English D type adRP pedigree from which only a single patient was available for analysis, a bandshift in the exon 5 PCR was found to be due to the codon 347 CCG to CTG (Pro to Leu) mutation first reported in the USA18 (data not shown). The remaining three families are English in origin and have been classified as having sectorial RP. Interestingly, all were found to have mutations in exon 1. Family adRP36 snowed a bandshift with both of the exon 1 primer sets and when sequenced was found to have a codon 58 ACG to AGG (Thr to Arg) mutation, as shown in figure 3. This mutation was also first described in US patients18. Family adRP48, from which again only a single affected individual was available, also proved to have this mutation (data not shown). Pedigree adRP37 revealed a bandshift only between primers A and K, due to a codon 106 mutation of GGG to AGG (Gly to Arg), as shown in figure 4. This can
Figure 2. Sequence from exon 1 of the rhodopsin gene in an affected member of family adRP28. Sequence was generated from a PCR of primers R against C (767bp), using primer A, and shows the mutation on the coding strand (CCC-CGC).
Table 1. Oligonucleotide primer sequences, written 5' to 3'. Numbers in brackets denote the position of the 5' base of the oligomer in the original sequence34. A: B: C: E: F:
G: I: K: L: M: N: O: P:
Q: R:
T C A T T T G G C G A G A G G
T A C T C C A C C C C A G G G
C T T G C A G T G T A A T A G
G G C G A C T A C T G G T T A
C T T C G G A
G C C A A
C G
C
A T C T A G G G T T A T C G A
G T C G C
C C T G T G G A G G
C C
c T C T T T C C G T A A A
A T A T A C T G T C C A G G C
T G G C T T G A G
C A G C A A
T C A C G G T G A T G A A C A
C T C C G A C C
c T A A C G G
T G C A C G C A T C G T A C T
T A
C A T G T G G T A G C C C
G T C G C G T G C G A C T T A
G C T T C T G A C C G C
G A T
G G C C T C G T T T G A T T G
T T C C C C C G T C A C
G A C
G
G A C C A A T G A G G G G A
G C T T A G G A C G G C G T G
(241)
mm (717)
A T
(3758) (4073) (4002) (4404) (533) G (2416) G (2636) (4126) (4226) (5111) (5322) (-50)
Human Molecular Genetics, Vol. 1, No. 1 43 be detected as loss of an Apal restriction site in PCR amplified rhodopsin exon 1. Families for which rhodopsin mutations have been found in this study are listed in table 2. These mutations are marked on a schematic representation of the rhodopsin molecule, shown in figure 5, together with those published in the US. The screen alsorevealedone further bandshift in family adRP6. However, this did not segregate with the adRP phenotype and on sequencing was found to be a neutral mutation changing GGC to GGT at codon 120 leaving the glycine residue unchanged. In order to estimate the frequency of such changes and as controls
OC \ A C T C C T T C A A C T
Figure 3. Sequence from rhodopsin exon 1 in a patient from adRP36, generated from a PCR of primers R against C, using primer A. The mutation is seen on the coding strand (ACG to AGG). °
DISCUSSION We have found mutations in the rhodopsin coding sequence in twelve of the 39 families screened in this study. Nine mutations were found in all, three of which were in two families each, while the rest occurred in single families. Of these, seven are mutations seen only in this study, while two were also observed in American patients18. From these results we obtain an estimate of approximately 30% for the proportion of adRP cases which result from rhodopsin mutations. However, the screen described does have limitations. Firstiy, mutations which deleted all or a substantial part of an oligomer used to PCR amplify would result in selective amplification of the normal allele only, so that the abnormality would not be detected. The oligomers selected lie well outside the exons, and such large scale deletion/insertion events might be expected to be relatively rare. Nevertheless, we have screened patients from all of our families by Southern blotting with frequent cutting enzymes, and have found no large deletions. Secondly, mutations causing adRP may lie outside the coding sequence, in introns or in the promoter region. Rhodopsin appears to have at least two upstream promoter sequences26. Both of diese have been screened for mutations in the panel, but none were observed. Thirdly the method used for the screen is novel and as yet has not been widely tested. While the results of this study demonstrate its ability to detect a range of mutations, it remains possible that a subset of mutations are undetected. However, since four of four controls were detected in the original test, it is probable that most if not all of the mutations in the panel have been detected. The method is rapid and non-radioactive and therefore provides a simple alternative to SSCP or HOT mutation detection techniques. Clinically, the twelve families with rhodopsin RP identified by this study consist of four with D-type RP, three with sectorial RP and five unclassified families. In contrast, six R-type families reported by this and other laboratories have shown significant exclusion of linkage to 3q marker C17 (D3S47), suggesting that the causative mutations in most, if not all R-type adRP derive from elsewhere in the genome14'15'27'28'29. However two of the six D-type families in the panel did not reveal a mutation in rhodopsin. Data from one of these families together with a large Irish pedigree has raised the possibility of a second D-type adRP
Table 2. Rhodopsin mutations found in the panel of ADRP families studied. Family
ADRP ADRP ADRP ADRP ADRP ADRP ADRP ADRP ADRP ADRP ADRP ADRP
Mutation
1 10 14 25 28 30 36 37 38 39 44 48
Reference
Codon
Sequence
Amino Acid Change
347 296 del 255 190 53 190 58 106 211 del 68-71 347 58
CCG-CTG AAG-GAG del ATC GAC-AAC CCC-CGC GAC-AAC ACG-AGG GGG-AGG CAC-CCC del CTGCG CACGCCT CCG-CTG ACG-AGG
Pro—Leu Lys-Glu delDe Asp— Asn Pro—Arg Asp—Asn Thr—Arg Gly-Arg His-Pro del Leu-Arg-Thr-Pro Pro—Leu Thr—Arg
Keen et al Keen et al Ingleheam Keen et al This study Keen et al This study This study Keen et al Keen et al This study This study
(1991a) (1991b) et al (1991) (1991b) (1991b) (1991b) (1991b)
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/
T G C A T C T C G b
to determine whether any neutral amino acid changes occurred in the normal population, 37 normal individuals were screened. However, no further bandshifts were observed.
44 Human Molecular Genetics, Vol. 1, No. 1
T
C A C C
populations in USA18, Great Britain (this study), Germany (Andreas Gal, personal communication) and Japan33, suggesting that mutations have arisen in different populations as separate events. However, in most instances each rhodopsin RP family appears to have a different mutation, making diagnosis and genetic counselling more complex. The finding of mutations in rhodopsin and the correlation of genotype with phenotype increases our understanding of the disease and of normal eye function and allows counselling for a number of UK adRP families. In addition, it may now be possible to define diagnostic criteria for differentiating between different forms of the disease, which would mean a more rapid molecular diagnosis, even in isolated cases with no family history. METHODS We have collected blood samples from a panel of 39 adRP families, the majority of which are British pedigrees from the Moorfields Eye Hospital RP register. In some families the pedigrees have been traced as far back as the early 18th century and ten of the pedigrees have proved large enough for independent genetic linkage analysis. Six have been classified as D-type and a further six as R-type using established criteria7. Three others have been designated as sectorial RP on the basis that all members of each family have altitudinal distribution of disease whatever their age, implying lack of progression. The remainder are unclassified to date. Genomic DNAs were prepared from patients' peripheral blood lymphocytes. Also, a set of primers were synthesized which could be used to amplify by polymerase chain reaction (PCR)23 the entire coding sequence of rhodopsin (Table 1 and Figure 1). The primers selected were 20—22 bp long with minimal secondary structure and a dissociation temperature of 60°C or above. They were derived from intronic sequence 2 0 - 5 0 bp out from the end of each rhodopsin exon, so that the exons and the immediately adjacent intron sequence could be amplified and screened. These were then used to prime the amplification of each exon in patients from each adRP family and 37 normal controls. PCR was performed in a 50/d reaction with 1/xg of genomic DNA, 20 pmoles of each primer, 200jiM dNTPs and 1 unit of Taq DNA polymerase (NBL) in buffer provided by the manufacturer. A two stage PCR with 30 cycles of 94°C for 30 seconds and 60°C for 4 minutes was found to be a reliable compromise programme which worked well with most primer sets. In the case of exon 2, annealing/extension was carried out at 67°C to prevent the presence of a persistent ghost band immediately above the main product. The PCR products generated were run on hydrolink D5000 gels (AT Biochem). This gel matrix has been demonstrated to resolve mutations as heteroduplexed
c
G G G C T T C T G C T T IHTRADtSCAl. DOMAIN
Figure 4. Sequence from exon 1 of the rhodopsin gene in an adRP37 patient, generated from the PCR product of primers A and C (477bp) using primer B. The mutation is shown on the coding strand (GGG to AGG).
Figure 5. Graphic representation of the rhodopsin molecule, with mutations found in this screen shaded.
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locus in the 3q region30. That all three families with sectorial adRP have mutations in rhodopsin exon 1 implies that a subgroup of rhodopsin mutations in this region cause this characteristic distribution of photoreceptor function loss. Furthermore, the codon 23 mutation, also in exon 1, has also been described as having a sectorial RP phenotype12 although the same paper describes the problems in defining diagnostic criteria for sectorial RP, and another US group finds a wide range of phenotypes in patients with the codon 23 mutation31. Our results have demonstrated a difference in severity of RP with different rhodopsin mutations32. However the phenotypes produced by the mutations described to date all fall into the categories of D-type RP and sectorial disease, while all families excluded by linkage are R-type. This accords with the view that D-type and R-type adRP have different pathogenetic mechanisms7. The ratio of rhodopsin to non-rhodopsin adRP observed in this study approximates to the ratio of D- to R-type adRP families noted by clinicians which is between 1 to 3 and 1 to 4 7 ' 8 . Therefore it seems possible that the D- and R-type clinical categories of adRP correspond to the underlying rhodopsin/non-rhodopsin genetic categories. The observations made in this and other studies give an insight into the population dynamics of a dominant genetic disease. The relatively late onset of total blindness in RP might be expected to reduce selection against it to a point where a causative mutation could persist in the population for several generations. Such a founder effect seems likely to explain the relatively high frequency of the codon 23 mutation in US RP patients. On the other hand the commoner codon 347 mutation has been observed in disparate
Human Molecular Genetics, Vol. 1, No. 1 45 DNA fragments with a shifted mobility relative to the homoduplexed normal product24. Gels of 24cm in length and lmm thick were prepared according to manufacturer's instructions. They were run over-night at room temperature for approximately 1700-2000 volt hours. Gels were then stained with ethidium bromide and photographed. Having identified exons which contained mutations, these were sequenced with sequenase (USB) primed from a kinase labelled oligomer by a method described previously23.
ACKNOWLEDGEMENTS We are grateful to the Special Trustees of the Royal Victoria Infirmary, Newcastle upon Tyne for supporting T.J.Keen and to the Wellcome Trust (Grant no. 18468/1.5/DG), National Retinitis Pigmentosa Foundation Fighting Blindness USA, the George Gund Foundation, the British Retinitis Pigmentosa Society and Newcastle University Research Committee for funding this research. Thanks also to our colleagues Professor Barrie Jay and Mr Tony Moore for patient details and for providing clinical samples and to Brenda Lauffart for excellent technical assistance. We are indebted to Pauline Battista and Rachelle Townsley for typing this manuscript.
REFERENCES
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1. Krill A£(1972). Retinitis pigmentosa: A review. Sight Sav. Rev. 42: 21-28. 2. Botermans CHG (1972). Primary pigmentary retinal degeneration and its association with neurological diseases. In Handbook of Clinical Neurology 13 (eds Vinken PJ and Bruyn GW) 148-379. 3. Bundey S and Crews SJ (1984). A study of retinitis pigmentosa in the City of Birmingham II. Clinical and genetic heterogeneity. J Med Genet 21: 421-428. 4. Berson EL, Gouras P, Gunkel RD and Myrianthopoulos NC (1969). Dominant Retinitis Pigmentosa with reduced penetrance. Arch Ophthalmol 89: 226-234. 5. Massof RW and Finkelstein D (1979). Rod sensitivity relative to cone sensitivity in retinitis pigmentosa. Invest Ophthalmol Visual Sci 18: 263-272. 6. Massof RW and Finkelstein D (1981). Two forms of autosomal dominant retinitis pigmentosa. Doc Ophthalmol 51: 289-346. 7. Lyness AL, Ernst W, Quinlan MP, Clover GM, Arden GB, Carter RM, Bird AC and Parker JA (1985) A clinical, psychophysical and electroretinographic survey of patients with autosomal dominant retinitis pigmentosa. Brit J Ophthalmol 69: 326-339 8. Farber MD, Fishman GA and Weiss RW (1985). Autosomal dominantly inherited retinitis pigmentosa: Visual acuity loss by subtype. Arch Ophthalmol 103: 524-528. 9. Berson EL and Howard J (1971). Temporal aspects of the electroretinogram in sector retinitis pigmentosa. Arch Ophthalmol 86: 653—665. 10. Massof RW and Finkelstein D (1979). Vision threshold profiles in sector retinitis pigmentosa. Arch Ophthalmol 91: 191-198. 11. Fulton AB and Hansen RM (1988). The relations of rhodopsin and scotopic retinal sensitivity in sector retinitis pigmentosa. Am J Ophthalmol 105: 132-140. 12. Heckenlively JR, Rodriguez JA and Daiger SP (1991). Autosomal Dominant Sectoral Retinitis Pigmentosa. Arch Ophthalmol 109: 84-90. 13. McWilliam P, Farrar GJ, Kenna P, Bradley DG, Humphries MM, Sharp EM, McConnell DJ, Lawler M, Sheils D, Ryan C, Stevens K, Daiger SP and Humphries P (1989). Autosomal dominant retinitis pigmentosa (ADRP): Localisation of an ADRP gene to the long arm of chromosome 3. Genomics 5: 619-622. 14. Lester DH, Inglehearn CF, Bashir R, Ackford H, Esakowitz L, Jay M, Bird AC, Wright AF and Bhattacharya SS. (1990). Linkage to D3S47 (C17) in one large family and exclusion in another: Confirmation of genetic heterogeneity. Am J Hum Genet. 47: 536-541. 15. Inglehearn CF, Jay M, Lester DH, Bashir R, Jay B, Bird AC, Wright AF, Evans HJ, Papiha SS and Bhattacharya SS (1990). No evidence for linkage between late onset autosomal dominant Retinitis Pigmentosa and Chromosome 3 locus D3S47 (C17). Evidence for genetic heterogeneity. Genomics 6: 168-173 16. Dryja TP, McGee TL, Reichel E, Hahn LB, Cowley GS, Yandell DW, Sandberg MA and Berson EL (1990). A point mutation of the rhodopsin gene in one form of retinitis pigmentosa. Nature 343: 364-366. 17. Basinger S, Bok D and Hall H (1976). Rhodopsin in the rod outer segment plasma membrane. J Cell Biol 69: 29-42. 18. Dryja TP, McGee TL, Hahn LB, Cowley GS.OUsonJE, Reichel E, Sandberg MA and Berson EL (1990). Mutations within the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa. New Engl J Medicine. 323: 1302-1307.
19. Dryja TP, McGee TL, Hahn GS, Cowley GS, OUson JE, Reichel E, Sandberg MA, and Berson EL (1990). Missense mutations within the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa. Abstract 207. American Society of Human Genetics Conference Ohio. Am J Hum Genet 47 (Supplement): 54. 20. Farrar GJ, Kenna P, Redmond R, McWilliam P, Bradley DG, Humphries M, Sharp EM, Inglehearn CF, Bashir R, Jay M, Watty A, Ludwig M, Schinzel A, Sammans AC, Gal A, Bhattacharya SS and Humphries P (1990). Autosomal dominant retinitis pigmentosa: Absence of the rhodopsin codon 23 prokne-histidine substitution in pedigrees of European origin. Am. J. Hum. Genet., 47, 941-945. 21. Inglehearn CF, Bashir R, Lester DH, Jay M, Bird AC and Bhattacharya SS (1991). A three basepair deletion in the rhodopsin gene in a family with autosomal dominant retinitis pigmentosa. Am J Hum Genet 48: 26-30. 22. Keen TJ, Inglehearn CF, Lester DH, Bashir R, Jay M, Bird AC, Jay B and Bhattacharya SS (1991). Autosomal dominant retinitis pigmentosa: four new mutations in rhodopsin, one of them in the retinal attachment she. Genomics. 11, 199-205. 23. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higushi R, Horn GT, Mullis KB and Erlich HA (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.Science 239: 487-91. 24. Keen J, Lester DH, Inglehearn CF, Curtis A and Bhattacharya SS. (1991). Rapid detection of single base mismatches as heteroduplexes on hydrolink gels. Trends Genet 7:5. 25. Yandell DW and Dryja TP. (1989) Detection of DNA sequence polymorphisms by enzymatic amplification and direct genomic sequencing. Am J Hum Genet 45: 547-555. 26. Zack DJ, Bennet J, Wang Y, Davenport C, Klaunberg B, Gearhart J and Nathans J (1991). Unusual topography of Bovine rhodopsin promotor-LacZ fusion gene expression in transgenic mouse retinas. Neurone 6: 187-99. 27. Farrar GJ, McWilliam P, Bradley DG, Kenna P, Lawler M, Sharp EM, Humphries MM, Eiberg H, Conneally PM, Trofatter JA, Humphries P (1990). Autosomal Dominant Retinitis Pigmentosa: Linkage to Rhodopsin and evidence for genetic heterogeneity. Genomics 8: 35—40. 28. Blanton SH, Cottingham AW, Giesenschlag N, Heckenlively JR, Humphries P and Daiger SP (1990). Further evidence of exclusion of linkage between type II Autosomal Dominant Retinitis Pigmentosa (ADRP) and D3S47 on 3q. Genomics 8: 179-181. 29. Kaplan J, Guasconi G, Dufier JL, Michel-Awad A, David A, Munnich A and Frezal J (1991). Exclusion of linkage between D3S47 (C17) and ADRP II gene in two large families of moderate autosomal dominant retinitis pigmentosa. evidence for genetic heterogeneity. Annales de Genctique 33: 152-154. 30 Inglehearn CF, Lester DH, Bashir R, Atif U, Keen TJ, Sertedaki A, Lindsey J, Jay M, Bird AC, Farrar GJ, Humphries P and Bhattacharya SS (1992). Recombination between rhodopsin and locus D3S47 (C17) in Rhodopsin retinitis pigmentosa families. Am J Hum Genet. In Press. 31. Berson EL, Rosner B, Sandberg MA and Dryja TP (1991). Ocular findings in patients with autosomal dominant Retinitis Pigmentosa and a Rhodopsin gene defect (Pro-23-His). Arch Ophthalmol 109: 92-101. 32. Fitzke FW, Owens S, Moore AT, Jay M, Inglehearn CF, Bhattacharya and Bird AC (1991). Comparison of Functional Characteristics of Autosomal Dominant Retinitis Pigmentosa with different Amino Acid changes in the rhodopsin molecule. ARVO Abstracts 1200 p912. 33. Fujiki K, Hotta Y, Shkmo T, Hayakawa M, Hashimoto T, Noro M, Sakuma T, Tamai M, Nakajima A and Kanai A (1991). Codon 347 mutation of the rhodopsin gene in a Japanese family with autosomal dominant Retinitis Pigmentosa. Invest Ophthalmol Vis Sci 32(Suppl): 1137. 34. Nathans J and Hogness DS (1984. Isolation and nucleotide sequence of the gene encoding human rhodopsin. Proc Nat! Acad Sci USA 81: 4851-4855
Human Molecular Genetics, Vol. 1, No. 1 41—45
A completed screen for mutations of the rhodopsin gene in a panel of patients with autosomal dominant retinitis pigmentosa Chris F.lngleheam, T.Jeffrey Keen, Rumaisa Bashir, Marcelle Jay2, Fred Fitzke2, Alan C.Bird2, Alex Crombie1 and Shomi Bhattacharya* Department of Human Genetics, department of Ophthalmology, University of Newcastle upon Tyne and department of Clinical Ophthalmology, Moorfields Eye Hospital, London, UK Received November 15, 1991; Revised and Accepted December 16, 1991
ABSTRACT
INTRODUCTION Retinitis pigmentosa (RP) is a term used to denote a number of inherited disorders causing progressive loss of peripheral vision and night-blindness, in many cases leading to blindness in later life. Classically they are characterised by bone-spicule like pigmentation observed in the mid-periphery of the retina in later stages of the disease process. Other clinical features include narrowed retinal vessels and a severely altered or non-recordable rod electro-retinogram (ERG)1. RP can be inherited in X-linked, dominant, and recessive forms, and is also found in syndromes with other phenotypic defects2. The autosomal dominant class (adRP) accounts for around 22% of RP patients3. Attempts have been made to sub-divide this category further on the basis of gene penetrance4, age of onset5'6 and distribution of pigment in the affected retina7. Two such classification systems are currently in common usage with approximately corresponding categories. D-type ('diffuse') or * To whom correspondence should be addressed
type I adRP causes diffuse and severe loss of rod function, but cone function is retained until much later in the disease process. Patients consistently experience night-blindness within the first ten years of life, though pigmentary changes may not become evident until the third decade. In the D-type, ERG and psychophysical testing shows that rod function is abnormal over the whole fundus with relative preservation of cone function7. In R-type ('regional') or type 2 adRP the age of onset varies both between and within families. Indeed, some R-type families include individuals who appear asymptomatic yet have transmitted the disease to the next generation. The R-type disease causes 'regional' or patchy and equal loss of rod and cone function. Dtype disease is estimated to account for 22% of adRP 78 , with the bulk of the remainder being R type disease. Another rarer category of adRP is sectorial RP, which consistantly affects only one or two quadrants of the retina within a family, with the remaining retina left intact. It is evident that sectorial RP as described is also heterogeneous. Some authors have reserved the term for disease in which there is no progression into the spared segments and the ERG has a normal implicit time9. Others have used the term to denote non-progressive disease but with dysfunction in the unaffected regions10'1'. The term has also been used to include disease in which one part of the retina is affected earlier and more profoundly than the remainder, but which is progressive and eventually affects the whole visual field12. Linkage analysis in families with adRP has demonstrated that some have a genetic defect located on chromosome 3q13'14 while others do not". Soon after the report of a genetic linkage, a base substitution was identified in codon 23 of the rhodopsin gene, which maps to chromosome 3q, in 17 of 148 apparendy unrelated adRP patients in the USA16. Rhodopsin is the rod photopigment which initiates the visual transduction cascade. It is composed of seven helical transmembrane domains, bounded by cytoplasmic and intradiscal loops (see figure 5), and represents 80—90% of the constituent protein of the rod outer segment17. The same US laboratory has since reported a further three point mutations in adRP, two in codon 347 and one in codon 5818. One of the codon 347 mutations and the codon 58 mutation occurred in single families, while the other codon 347 mutation was found in 8 out of 150 adRP patients. However, haplotyping of probes from
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Recently it has been demonstrated that some families with autosomal dominant retinitis pigmentosa (adRP) have mutations in the rhodopsin gene while others do not. Previously we have identified six such mutations in seven adRP families in this laboratory, one of which was previously described in US patients. We now present a completed screen of the rhodopsin gene in a panel of 39 adRP families, by a rapid screening technique which will be of use for routine diagnosis. Nine different mutations were ultimately found, in a total of twelve of the 39 families. These include the six previously identified mutations, in codons 6 8 - 7 1 , 190, 211, 255, 296 and 347, two new ones in codons 53 and 106, and another mutation first identified in a single US patient, in codon 58. Thus approximately 30% of adRP families have 'Rhodopsin RP' while the remainder probably have a defect elsewhere in the genome. Of those families in which rhodopsin mutations have been found, four have been classified D type, three as sectorial RP and the remainder are of uncertain classification. All families excluded from chromosome 3q by linkage have been classified R type. These data suggest a correlation between clinical sub-classification and the underlying rhodopsin/non-rhodopsin heterogeneity.
42 Human Molecular Genetics, Vol. 1, No. 1 around the rhodopsin gene region suggested that individuals with the codon 23 mutation may have a common ancestor, as was the case in patients with the commoner codon 347 mutation19. A screen of some 91 European adRP families revealed none with a codon 23 mutation20. We have described previously five new rhodopsin mutations and also observed the commoner codon 347 mutation in European adRP patients2122. These were observed in a panel of 39 adRP families for which genealogical evidence is available to demonstrate that they are not related in recent generations. We now present a screen of the entire coding sequence of rhodopsin in patients from every family on that panel. The results obtained give an estimate for the frequency of rhodopsin RP as a fraction of total adRP. In addition, the completed screen demonstrates an apparent correlation between phenotype and genotype in adRP, and provides an insight into the population dynamics of a dominant genetic condition.
Figure 1. A graphic representation of the genomic structure of rhodopsin, drawn approximately to scale. Hatched areas represent exons, which are numbered 1 to 5 reading from 5' to 3'. Oligomers used in this study are shown as lines above (coding strand) and below (non-coding strand) the graphic, and are assigned letters which correspond to those shown in table 1.
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RESULTS Of the families screened in this project, seven have already been reported to have mutations in rhodopsin. These consist of families with a codon 347 point mutation24, a deletion of codon 25521, point mutations in codons 190 (two families), 211 and 296, and a deletion of codons 68-71 2 2 . In completing the screen, we have identified mutations in a further five families. In adRP 28, a small clinically unclassified pedigree of Scottish origin, a bandshift was identified between primers A and K in exon 1. When sequenced, this was found to be due to a CCC to CGC (Pro to Arg) mutation in codon 53 (Figure 2). In family adRP 44, an English D type adRP pedigree from which only a single patient was available for analysis, a bandshift in the exon 5 PCR was found to be due to the codon 347 CCG to CTG (Pro to Leu) mutation first reported in the USA18 (data not shown). The remaining three families are English in origin and have been classified as having sectorial RP. Interestingly, all were found to have mutations in exon 1. Family adRP36 snowed a bandshift with both of the exon 1 primer sets and when sequenced was found to have a codon 58 ACG to AGG (Thr to Arg) mutation, as shown in figure 3. This mutation was also first described in US patients18. Family adRP48, from which again only a single affected individual was available, also proved to have this mutation (data not shown). Pedigree adRP37 revealed a bandshift only between primers A and K, due to a codon 106 mutation of GGG to AGG (Gly to Arg), as shown in figure 4. This can
Figure 2. Sequence from exon 1 of the rhodopsin gene in an affected member of family adRP28. Sequence was generated from a PCR of primers R against C (767bp), using primer A, and shows the mutation on the coding strand (CCC-CGC).
Table 1. Oligonucleotide primer sequences, written 5' to 3'. Numbers in brackets denote the position of the 5' base of the oligomer in the original sequence34. A: B: C: E: F:
G: I: K: L: M: N: O: P:
Q: R:
T C A T T T G G C G A G A G G
T A C T C C A C C C C A G G G
C T T G C A G T G T A A T A G
G G C G A C T A C T G G T T A
C T T C G G A
G C C A A
C G
C
A T C T A G G G T T A T C G A
G T C G C
C C T G T G G A G G
C C
c T C T T T C C G T A A A
A T A T A C T G T C C A G G C
T G G C T T G A G
C A G C A A
T C A C G G T G A T G A A C A
C T C C G A C C
c T A A C G G
T G C A C G C A T C G T A C T
T A
C A T G T G G T A G C C C
G T C G C G T G C G A C T T A
G C T T C T G A C C G C
G A T
G G C C T C G T T T G A T T G
T T C C C C C G T C A C
G A C
G
G A C C A A T G A G G G G A
G C T T A G G A C G G C G T G
(241)
mm (717)
A T
(3758) (4073) (4002) (4404) (533) G (2416) G (2636) (4126) (4226) (5111) (5322) (-50)
Human Molecular Genetics, Vol. 1, No. 1 43 be detected as loss of an Apal restriction site in PCR amplified rhodopsin exon 1. Families for which rhodopsin mutations have been found in this study are listed in table 2. These mutations are marked on a schematic representation of the rhodopsin molecule, shown in figure 5, together with those published in the US. The screen alsorevealedone further bandshift in family adRP6. However, this did not segregate with the adRP phenotype and on sequencing was found to be a neutral mutation changing GGC to GGT at codon 120 leaving the glycine residue unchanged. In order to estimate the frequency of such changes and as controls
OC \ A C T C C T T C A A C T
Figure 3. Sequence from rhodopsin exon 1 in a patient from adRP36, generated from a PCR of primers R against C, using primer A. The mutation is seen on the coding strand (ACG to AGG). °
DISCUSSION We have found mutations in the rhodopsin coding sequence in twelve of the 39 families screened in this study. Nine mutations were found in all, three of which were in two families each, while the rest occurred in single families. Of these, seven are mutations seen only in this study, while two were also observed in American patients18. From these results we obtain an estimate of approximately 30% for the proportion of adRP cases which result from rhodopsin mutations. However, the screen described does have limitations. Firstiy, mutations which deleted all or a substantial part of an oligomer used to PCR amplify would result in selective amplification of the normal allele only, so that the abnormality would not be detected. The oligomers selected lie well outside the exons, and such large scale deletion/insertion events might be expected to be relatively rare. Nevertheless, we have screened patients from all of our families by Southern blotting with frequent cutting enzymes, and have found no large deletions. Secondly, mutations causing adRP may lie outside the coding sequence, in introns or in the promoter region. Rhodopsin appears to have at least two upstream promoter sequences26. Both of diese have been screened for mutations in the panel, but none were observed. Thirdly the method used for the screen is novel and as yet has not been widely tested. While the results of this study demonstrate its ability to detect a range of mutations, it remains possible that a subset of mutations are undetected. However, since four of four controls were detected in the original test, it is probable that most if not all of the mutations in the panel have been detected. The method is rapid and non-radioactive and therefore provides a simple alternative to SSCP or HOT mutation detection techniques. Clinically, the twelve families with rhodopsin RP identified by this study consist of four with D-type RP, three with sectorial RP and five unclassified families. In contrast, six R-type families reported by this and other laboratories have shown significant exclusion of linkage to 3q marker C17 (D3S47), suggesting that the causative mutations in most, if not all R-type adRP derive from elsewhere in the genome14'15'27'28'29. However two of the six D-type families in the panel did not reveal a mutation in rhodopsin. Data from one of these families together with a large Irish pedigree has raised the possibility of a second D-type adRP
Table 2. Rhodopsin mutations found in the panel of ADRP families studied. Family
ADRP ADRP ADRP ADRP ADRP ADRP ADRP ADRP ADRP ADRP ADRP ADRP
Mutation
1 10 14 25 28 30 36 37 38 39 44 48
Reference
Codon
Sequence
Amino Acid Change
347 296 del 255 190 53 190 58 106 211 del 68-71 347 58
CCG-CTG AAG-GAG del ATC GAC-AAC CCC-CGC GAC-AAC ACG-AGG GGG-AGG CAC-CCC del CTGCG CACGCCT CCG-CTG ACG-AGG
Pro—Leu Lys-Glu delDe Asp— Asn Pro—Arg Asp—Asn Thr—Arg Gly-Arg His-Pro del Leu-Arg-Thr-Pro Pro—Leu Thr—Arg
Keen et al Keen et al Ingleheam Keen et al This study Keen et al This study This study Keen et al Keen et al This study This study
(1991a) (1991b) et al (1991) (1991b) (1991b) (1991b) (1991b)
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/
T G C A T C T C G b
to determine whether any neutral amino acid changes occurred in the normal population, 37 normal individuals were screened. However, no further bandshifts were observed.
44 Human Molecular Genetics, Vol. 1, No. 1
T
C A C C
populations in USA18, Great Britain (this study), Germany (Andreas Gal, personal communication) and Japan33, suggesting that mutations have arisen in different populations as separate events. However, in most instances each rhodopsin RP family appears to have a different mutation, making diagnosis and genetic counselling more complex. The finding of mutations in rhodopsin and the correlation of genotype with phenotype increases our understanding of the disease and of normal eye function and allows counselling for a number of UK adRP families. In addition, it may now be possible to define diagnostic criteria for differentiating between different forms of the disease, which would mean a more rapid molecular diagnosis, even in isolated cases with no family history. METHODS We have collected blood samples from a panel of 39 adRP families, the majority of which are British pedigrees from the Moorfields Eye Hospital RP register. In some families the pedigrees have been traced as far back as the early 18th century and ten of the pedigrees have proved large enough for independent genetic linkage analysis. Six have been classified as D-type and a further six as R-type using established criteria7. Three others have been designated as sectorial RP on the basis that all members of each family have altitudinal distribution of disease whatever their age, implying lack of progression. The remainder are unclassified to date. Genomic DNAs were prepared from patients' peripheral blood lymphocytes. Also, a set of primers were synthesized which could be used to amplify by polymerase chain reaction (PCR)23 the entire coding sequence of rhodopsin (Table 1 and Figure 1). The primers selected were 20—22 bp long with minimal secondary structure and a dissociation temperature of 60°C or above. They were derived from intronic sequence 2 0 - 5 0 bp out from the end of each rhodopsin exon, so that the exons and the immediately adjacent intron sequence could be amplified and screened. These were then used to prime the amplification of each exon in patients from each adRP family and 37 normal controls. PCR was performed in a 50/d reaction with 1/xg of genomic DNA, 20 pmoles of each primer, 200jiM dNTPs and 1 unit of Taq DNA polymerase (NBL) in buffer provided by the manufacturer. A two stage PCR with 30 cycles of 94°C for 30 seconds and 60°C for 4 minutes was found to be a reliable compromise programme which worked well with most primer sets. In the case of exon 2, annealing/extension was carried out at 67°C to prevent the presence of a persistent ghost band immediately above the main product. The PCR products generated were run on hydrolink D5000 gels (AT Biochem). This gel matrix has been demonstrated to resolve mutations as heteroduplexed
c
G G G C T T C T G C T T IHTRADtSCAl. DOMAIN
Figure 4. Sequence from exon 1 of the rhodopsin gene in an adRP37 patient, generated from the PCR product of primers A and C (477bp) using primer B. The mutation is shown on the coding strand (GGG to AGG).
Figure 5. Graphic representation of the rhodopsin molecule, with mutations found in this screen shaded.
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locus in the 3q region30. That all three families with sectorial adRP have mutations in rhodopsin exon 1 implies that a subgroup of rhodopsin mutations in this region cause this characteristic distribution of photoreceptor function loss. Furthermore, the codon 23 mutation, also in exon 1, has also been described as having a sectorial RP phenotype12 although the same paper describes the problems in defining diagnostic criteria for sectorial RP, and another US group finds a wide range of phenotypes in patients with the codon 23 mutation31. Our results have demonstrated a difference in severity of RP with different rhodopsin mutations32. However the phenotypes produced by the mutations described to date all fall into the categories of D-type RP and sectorial disease, while all families excluded by linkage are R-type. This accords with the view that D-type and R-type adRP have different pathogenetic mechanisms7. The ratio of rhodopsin to non-rhodopsin adRP observed in this study approximates to the ratio of D- to R-type adRP families noted by clinicians which is between 1 to 3 and 1 to 4 7 ' 8 . Therefore it seems possible that the D- and R-type clinical categories of adRP correspond to the underlying rhodopsin/non-rhodopsin genetic categories. The observations made in this and other studies give an insight into the population dynamics of a dominant genetic disease. The relatively late onset of total blindness in RP might be expected to reduce selection against it to a point where a causative mutation could persist in the population for several generations. Such a founder effect seems likely to explain the relatively high frequency of the codon 23 mutation in US RP patients. On the other hand the commoner codon 347 mutation has been observed in disparate
Human Molecular Genetics, Vol. 1, No. 1 45 DNA fragments with a shifted mobility relative to the homoduplexed normal product24. Gels of 24cm in length and lmm thick were prepared according to manufacturer's instructions. They were run over-night at room temperature for approximately 1700-2000 volt hours. Gels were then stained with ethidium bromide and photographed. Having identified exons which contained mutations, these were sequenced with sequenase (USB) primed from a kinase labelled oligomer by a method described previously23.
ACKNOWLEDGEMENTS We are grateful to the Special Trustees of the Royal Victoria Infirmary, Newcastle upon Tyne for supporting T.J.Keen and to the Wellcome Trust (Grant no. 18468/1.5/DG), National Retinitis Pigmentosa Foundation Fighting Blindness USA, the George Gund Foundation, the British Retinitis Pigmentosa Society and Newcastle University Research Committee for funding this research. Thanks also to our colleagues Professor Barrie Jay and Mr Tony Moore for patient details and for providing clinical samples and to Brenda Lauffart for excellent technical assistance. We are indebted to Pauline Battista and Rachelle Townsley for typing this manuscript.
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