GENOMICS
6.168-173
(1990)
No Evidence for Linkage between Late Onset Autosomal Dominant Retinitis Pigmentosa and Chromosome 3 Locus D3S47 (Cl 7): Evidence for Genetic Heterogeneity C. F. INGLEHEARN,* “Molecular
M. JAy,t D. H. LESTER,* R. BASHIR,* B. JAY,t A. C. BIRD,t A. F. WRIGHT,* H. J. EVANS,* S. S. PAPIHA,* AND 5. S. BHATTACHARYA**’
Genetics Unit, Human Genetics, University of Newcastle upon Tyne; tDepartment of Clinical Ophthalmology, Moorfields Eye Hospital, City Road, London; and $MRC Human Genetics Unit, Western General Hospital, Edinburgh, United Kingdom Received
September
18, 1989;
INTRODUCTION
Retinitis pigmentosa (RP) is one of the most common human inherited eye disorders and accounts for approximately 7% of blindness in the United Kingdom (DHSS, 1979). Typically, patients complain of night blindness and progressive narrowing of the visual field. Clinically it is characterized in later stages by bonespiculelike pigmentation in the midperiphery of the retina and a severely attenuated or nonrecordable rod electroretinogram (reviewed by Krill, 1972). RP is familial and occurs in X-linked, autosomal recessive, and autosomal dominant forms. The autosomal dominant category represents approximately 25% of total RP in the British population (Bundey and Crews, 1984). There is substantial variation in prognosis within this grouping, suggesting an underlying genetic heterogeneity. Berson et al. (1969) suggestedthat families could 1 TO whom correspondence should be addressed at University of Newcastle upon Tyne, Molecular Genetics Unit, Upper Claremont Street, Newcastle upon Tyne NE2 4AJ, UK.
$3.ocl
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
October
24, 1989
be classified on the basis of the degree of gene penetrance. A further classification put forward by Massof and Finkelstein (1979) subdivided ADRP on the basis of differential effects on rod relative to cone sensitivity of long- and short-wavelength stimuli. The same authors also suggested a division based on age of onset: type I (early onset) with night blindness before 10 years, and type II (late onset) beginning in the third decade (Massof and Finkelstein, 1981). More recently ADRP has been subdivided on the basis of persistence of a measurable rod electroretinogram (Arden et al., 1983) and also on distribution of pigmentation in an affected retina in the early stages of the disease (Lyness et al., 1985; Farber et al., 1985). A classification on the latter basis gives the diffuse (D) and regional (R) types, which correspond to the early (type I) and late (type II) onset categories of Massof and Finkelstein (1981), respectively. Variation in the disease status within these groups and even within families makes it impossible, however, to determine whether these clinical categories correspond to different genetic lesions. Use of a linked genetic marker would make it possible to determine whether these differences are allelic or result from defects in different genes. Markers that are closely linked to the ADRP phenotype within families would also be valuable tools to genetic counselors, allowing more accurate detection of gene carriers and of disease status in prenatal diagnoses. Ultimately, close genetic linkage may also lead to isolation of the defective gene itself, which would increase an understanding of the molecular pathology of this complex disease and may facilitate therapeutic intervention at a presymptomatic stage. The first recorded linkage study in ADRP examined four extended pedigrees using a number of well-defined
Retinitis pigmentosa is an inherited form of blindness caused by progressive retinal degeneration. P. McWilliam et ul. (1989, Genomics 5: 619-622) demonstrated close genetic linkage between autosomal dominant retinitis pigmentosa (ADRP) and locus D3S47 (C17) in a single early onset pedigree. The marker Cl7 maps to the long arm of chromosome 3. Clinically, the disease phenotype has been subdivided into at least two forms on the basis of age of onset, as well as electrodiagnostic criteria. We demonstrate that Cl7 is unlinked in a late onset pedigree, indicating that the phenotypic variation seen reflects underlying genetic heterogeneity. 0 1~90 Academic Pre~e, Inc.
os&u3-7543/90
revised
168
LOCUS
HETEROGENEITY
IN
biochemical markers and pooling the results of the families in calculating lod scores (Hussels-Maumenee et al., 1975). No significant linkage was observed. Such an approach, however, would be flawed if genetic heterogeneity existed. A similar study, using only a single large pedigree (Spence et al., 1977), reported a maximum lod score of 2.5 at a recombination fraction of 0.10 to rhesus on chromosome 1. However, further data on the same family decreased that figure to 2.13 (Heckenlively et al., 1982). A study that pooled data from that pedigree and four others obtained a lod score of 1.51 between rhesus and ADRP, while all other biochemical markers tested gave significantly smaller lod scores (Field et aZ., 1982). Pooling of data from a further two families gave a final lod score of 1.89 to rhesus, still not reaching the generally accepted required value of 3 for statistical significance (Daiger et al., 1987). Results from a Chinese study further support a chromosome 1 linkage but remain unconfirmed (Yijian et al., 1987), while studies using DNA probes around rhesus have found no linkage in two large pedigrees (Bradley et al., 1989; Inglehearn et al., 1989). Recently, as a result of extensive linkage studies using the probes reported by Donis-Keller et al. (1987), McWilliam et al. (1989) have reported tight linkage between ADRP and a cosmid clone, C17, mapping to chromosome 3q in a single large type I (early onset) Irish pedigree. In this paper, we present results of a genetic linkage study between Cl7 and a large R type (type II) late onset pedigree with partial penetrance. Cl7 was found to be unlinked in this family, which confirms the existence of genetic heterogeneity within ADRP as suggested by clinical observations. MATERIALS
AND
METHODS
The approach of Don&-Keller et al. (1987) in constructing a complete linkage map of the human genome favored the use of whole cosmid and phage clones, since, when adequately competed, a large insert is more likely to detect a polymorphism. Probe Cl7 is a 40-kb insert isolated from an Mb01 partial digest library of the human genome constructed in cosmid vector c2RB (Collaborative Research Inc.). The cosmid clone was grown by alkaline lysis maxiprep, as described by Maniatis et al. (1982). Human genomic DNAs were prepared from lymphocytes obtained from lo-ml blood samples by the method described by Herman and Frischauf (1987). Restriction digestion was carried out in buffer supplied by the manufacturer (NBL), for between 4 and 16 h, to between 2 and 20 times the recommended required digestion. TaqI digests were carried out at 65”C, while MspI digests appeared to work best at room temperatures (20-25 “C) as recommended by Collaborative Research Inc. Samples were size-fractionated on
AUTOSOMAL
DOMINANT
RP
169
1% agarose gels run overnight at 1.5 to 2 V/cm. Gels were denatured at high pH, neutralized, and then blotted to Hybond-N nylon membranes (Amersham) by the method of Southern (1975). DNA was fixed to the membranes by uv transillumination and then air-dried for 24 h. Filters were prehybridized in 0.5 M sodium phosphate buffer and 7% SDS for approximately 30 min and then hybridized in the same solution but with 10% dextran sulfate and radiolabeled probe. Whole cosmid Cl7 (50 ng) was oligo-labeled with [32P]dTTP by the method of Feinberg and Vogelstein (1983) and purified on a Sephadex G-50 column (Pharmacia) to remove unincorporated nucleotides. The labeled cosmid was then competitively hybridized to sheared total human DNA to reduce lane background caused by common repeats in the cosmid (Sealey et aZ., 1985). Labeled probe (50 ng) was competed against 4 mg total human DNA in 5X SSC in a total volume of 1.3 ml at 65°C for 30 min. The mix was then added to 20 ml of the hybridization solution. Filters were hybridized in polythene bags overnight at 65”C, rinsed in 2X SSC and 0.1% SDS at room temperature, and washed twice at 65°C in 0.5X SSC and 0.1% SDS. Autoradiographic images were obtained by exposure for between 1 and 4 days at -80°C to Kodak XAR5 film in Kodak regular cassettes. Two-point lod scores were calculated using the computer program LIPED (Ott, 1974). RESULTS
Polymorphisms Detected by Cl 7 Figure 1 shows panels of DNA from five unrelated individuals. The DNAs were digested with enzymes TaqI and MspI and then probed with competed C17. Twelve bands can be seen on an MspI digest. The top two bands, at 14 and 12.3 kb, are allelic. Frequencies of the upper (A) allele and lower (a) allele in 44 unrelated individuals were 0.35 and 0.65, respectively. Frequencies within family ADRP5 were 0.36 for A and 0.64 for a. This has proved to be the most informative allele system detected by this probe in ADRP5. Polymorphic bands can also be seen at approximately 8.7 and 6.7 kb, while weaker bands appear at approximately 5.5 and 3.5 kb. However, in some individuals, bands at 6.7 and 8.7 kb were either much weaker or totally absent, while the lower bands were correspondingly stronger (data not shown). The enzyme MspI recognizes the motif CCGG and cuts regardless of whether the second C is methylated. However, if this sequence is preceded by two guanine residues, to give GGCCGG, then the enzyme will cut only an unmethylated site (Nelson and McClelland, 1987). It is probable therefore that this second allele system is bounded on one side by such a site. In reactions sampled at time points of
170
INGLEHEARN
ET
Normal DNAs
could also be seen with this probe at approximately 3.2, 2.4, 2.3, 1.9, 1.2, 1.0, and 0.75 kb. In TuqI digests the Cl7 probe detects 13 bands. Again the upper two bands, at approximately 6.8 and 7.2 kb, are allelic. In 45 unrelated individuals the upper allele (T) was found to have a frequency of 0.17 and the lower (t) 0.83, while in the ADRP5 family, frequencies are 0.23 and 0.77, respectively. In samples that are visibly discernible as partial digests, an extra band can be seen at 6.3 kb. However, the band at 4.4 kb is clearly not the result of partial digestion and appears to represent one allele of a second polymorphism detected with this probe on Tag1 digests. Since the second allele could not be interpreted with certainty, this polymorphic system was not used in linkage analysis of ADRP. Constant bands in TuqI digests were seen at sizes of approximately 6.5, 4.2, 4.0, 3.2, 2.6, 2.3, 1.9, 1.7, 1.0, and 0.6 kb. Clinical
Cl7
increasing digestion, the lower bands were found to increase in strength but then stop at a point that varied in different individuals, suggesting differing degrees of methylation at this site (data not shown). Upper bands never disappeared completely in individuals tested in this way, although these bands had been seen to do so in other individuals. Because of the potential ambiguity in interpretation of this polymorphism, a lod score was not calculated for this system. Constant MspI bands
BP tt
2
l n 2.
Pedigree
Affected
ADRPB
0
showing
0
Unaffected
autosomal
Classification
of the Family
Studied
Figure 2 shows the complete pedigree known as ADRP5. It can be clearly seen that the pattern of inheritance in this family is autosomal dominant. By carrying out linkage studies only in a single large pedigree, we remove any problem associated with heterogeneity of disease locus in ADRP. Shaded symbols represent individuals affected with retinitis pigmentosa. However, the extent to which individuals are affected varies greatly. Onset of the defect may be at any time between 15 and 40 years of age, with night blindness as the first symptom. In some individuals the defect has led to complete loss of sight, although others retain good visual acuity until relatively late in life. In three
FIG. 1. DNA, MspI and TagI digested and probed with competed C17, from six unrelated individuals. Allelic bands used in the linkage analysis are indicated by arrowheads.
FIG.
AL.
@
dominant
q
::
saMk Tf
Unaffected below 40years
segregation
of the type
II late onset phenotype.
Tt
tt
Aa
Tt
Aa It
LOCUS
HETEROGENEITY
IN
AUTOSOMAL
DOMINANT
171
RP
ADRPS
1
2
3
4
5
6
7
FIG. 3. Genomic DNAs from ADRP5, MspI (upper) to individuals numbered on the pedigree above. Several yet16is homozygous AA and 20ishomozygous aa.
8
9 IO 11 12 13 14 15 16 17 18 19 20 21
and ToqI (lower) digested and probed with competed C17. Track numbers correspond crossovers can be seen; for example, tracks 16 and 20 have DNAs from affected sibs
cases, individuals with affected offspring, who were therefore obligate carriers, were so mildly affected that they were unaware of any problems. The only detectable defect in these patients was a slight elevation of rod thresholds on static perimetry testing as described by Ernst et al. (1983). To counter the problem of partial penetrance, we have used only data from affected individuals and from those above the age of 40, who were still unaffected. The pattern of pigmentation in the retinas of affected individuals and age of onset in this family are consistent with regional (R type) RP. Linkuge Analysis Genomic DNAs from the pedigree shown were digested with restriction enzymes TaqI and MspI, re-
TABLE Lod Scores
between
spectively, blotted, and probed with C17. Alleles for the upper MspI and upper TaqI systems for the entire family are shown in Fig. 2. Autoradiographs for part of the family highlighting a number of crossovers are shown in Fig. 3. Table 1 shows the results of two-point linkage analysis between ADRP and the two polymorphisms in this family. The number of informative meioses for the MspI polymorphism was larger than that for the TaqI polymorphism and this can be seen from the distribution of alleles at each of the two sites, as shown in Fig. 2. However, negative lod scores for both polymorphisms are still seen up to a recombination fraction of 0.30. Therefore, it is clear that both polymorphic sites identified by the probe Cl7 are unlinked to the ADRP phenotype in this family.
1
C17-Associated Restriction Fragment Length Polymorphisms Dominant Retinitis Pigmentosa Family Recombination
fraction
and an Autosomal
(0)
Main locus
Enzyme
0.000
0.001
0.05
0.10
0.20
0.30
ADRP ADRP
MspI Td
-
-12.593 -5.865
-3.917 -2.298
-2.265 -1.586
-0.772 -0.781
-0.178 -0.312
0.40
0.50
0.009 -0.086
0.000 0.000
172
INGLEHEARN DISCUSSION
The results presented here, considered together with those of McWilliam et al. (1989), prove that there is genetic heterogeneity in autosomal dominant retinitis pigmentosa. Further experiments underway in our laboratory should soon determine whether the Cl7 linkage demonstrated by McWilliam and co-workers holds true for all early onset (diffuse) ADRP families or whether there is further genetic variation within this group. In addition, in all the families that prove to be Cl7 unlinked, the task of searching for linkage will continue. It is interesting to note that Field et al. (1982) presented data that excluded an ADRP gene from nearly 40 CM around the transferrin (TF) locus, at 3q21. As yet, probe Cl7 has only been mapped relative to other probes from Collaborative Research and may therefore be some distance from TF, although their chromosomal locations appear to correspond. Alternatively, the exclusion of an ADRP locus from the region containing TF by Field and co-workers may have been due to the pooling of data from what we now know to be C17linked and unlinked families. Transferrin is a highly polymorphic genetic system and it will be of interest to analyze further the linkage relationships in C17linked ADRP families. In the short term, heterogeneity within ADRP families will limit the usefulness of the reported linkage in counseling and carrier diagnosis to those families that demonstrate close linkage with C17. The information on the use of the polymorphisms detected by Cl7 presented here should be of value to other researchers studying ADRP families, since the use of several polymorphic systems should make the probe informative in most pedigrees. ACKNOWLEDGMENTS We are grateful to the American Retinitis Pigmentosa Foundation and the George Gund Foundation for their generous support of this work. We thank Helen Ackford, Dawn Thistleton, and Alison Hardcastle for excellent technical support. Thanks also to Dr. Peter Humphries for informing us of his results before publication. Our special thanks to Dr. Anthony Moore for clinical assessment of members of this ADRP family. Finally, we are grateful to Pauline Battista for her patience in typing this manuscript. REFERENCES 1. ARDEN, G. B., CARTER, R. M., HOGG, C. R., POWELL, D. J., ERNST, W. J. K., CLOVER, G. M., LYNESS, A, L., AND QIJINLAN, M. P. (1983). Rod and cone activity in patients with dominantly inherited retinitis pigmentosa: Comparisons between psychophysical and electroretinographic measurements. &it. J. Ophthalmol. 67: 405-418. 2. BERSON, E. L., GOURAS, P., GUNKEL, R. D., AND MYRIANTHOPOULOS, N. C. (1969). Dominant retinitis pigmentosa with reduced penetrance. Arch. Ophthalmol. 99: 226-234.
ET AL. 3. BRADLEY, D. G., FARRAR, G. J., SHARP, E. M., KENNA, P., HUMPHRIES, M. M., MCCONNELL, D. J., DAIGER, S. P., MCWILLIAM, P., AND HUMPHRIES, P. (1989). Autosomal dominant retinitis pigmentosa: Exclusion of the gene from the short arm of chromosome 1 including the region surrounding the rhesus locus. Amer.
J. Hum.
Genet.
44:
570-576.
4. 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. 5. DAIGER, S. P., HECKENLIVELY, J. R., LEWIS, R. A., AND PELIAS, M. Z. (1987). DNA linkage studies in degenerative retinal diseases. In “Degenerative Retinal Diseases: Clinical and Laboratory Investigations” (J. G. Hollyfield and M. W. LaVail, Ii%.), pp. 147-162, A. R. Liss, New York. 6. DHSS (1979). Report on Public Health and Medical Subjects No. 129, Blindness and partial sight in England 1969-76, HMSO, London. 7. DONIS-KELLER, H., GREEN, P., HELMS, C., et al. (1987). A genetic linkage map of the human genome. Cell 57: 319-337. 8. ERNST, W., FAULKNER, D. J., HOGG, C. R., POWELL, D. J., ARDEN, G. B., AND VAEGAN (1983). An automated static perimeter/adaptometer using light emitting diodes. &it. J. Ophthulmol. 67:431-432. 9. FARBER, M. D., FISHMAN, G. A., AND WEISS, R. W. (1985). Autosomal dominantly inherited retinitis pigmentosa: Visual acuity loss by subtype. Arch. Ophthulmol. 103: 524-528. 10. FEINBERG, A. P., AND VOGELSTEIN, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13. 11. FIELD, L. L., HECHKENLIVELY, J. R., SPARKES, R. S., GARCIA, C. A., FARSON, C., ZEDALIS, D., SPAFXES, M. C., GRIST, M., TIDEMAN, S., AND SPENCE, M. A. (1982). Linkage analysis of five pedigrees affected with typical autosomal dominant retinitis pigmentosa. Amer. J. Med. Genet. 19: 266-270. 12. HECKENLIVELY, J. R., PEARLMAN, J. T., SPARKES, R. S., SPENCE, M. A., ZEDALIS, D., FIELD, L. L., SPARKES, M. C., CRIST, M., AND TIDEMAN, S. (1982). Possible assignment of a dominant retinitis pigmentosa gene to chromosome 1. Ophthalmic
Res. 14: 46-53.
13. HERMAN, B. G., AND FRISCHAUF,A. (1987). In “Guide to Molecular Cloning Techniques” (S. L. Berger and A. R. Kimmel, Eds.), pp, 180-183, Academic Press, London. 14. HUSSELS-MAUMENEE, I., PIERCE, E. R., BIAS, W. B., AND SCHLEUTERMANN, D. A. (1975). Linkage studies of typical retinitis pigmentosa and common markers. Amer. J. Hum. Genet. 27: 505-508. 15. INGLEHEARN, C. F., PAPIHA, S. S., JAY, M., MOORE, T., AND BHATTACHARYA, S. S. (1989). Linkage of internal minisatellite loci on chromosome 1 and exclusion of autosomal dominant retinitis pigmentosa proximal to rhesus. J. Med. Genet., in press. 16. KRILL, A. E. (1972). Retinitis pigmentosa: A review. Sight Sav. Rev. 42: 21-28. 17. LYNESS, A. L., ERNST, W., QUINLAN, M. P., GLOVER, G. M., ARDEN, G. B., CARTER, R. M., BIRD, A. C., AND PARKER, J. A. (1985). A clinical, psychophysical, and electroretinographic survey of patients with autosomal dominant retinitis pigmentosa. Brit. J. Ophthalmol. 69: 326-339. 18. MANIATIS, T., FRITSCH, E. F., AND SAMBROOK, J. (1982). “MOlecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 19. MASSOF, R. W., AND FINKELSTEIN, D. (1979). Rod sensitivity relative to cone sensitivity in retinitis pigmentosa. Invest. Ophthalmol. Visual Sci. 18: 263-272.
LOCUS
HETEROGENEITY
20. MASSOF, R. W., AND FINKELSTEIN, D. (1981). Two forms of autosomal dominant retinitis pigmentosa. Dot. Ophthuhnol. 61: 289-346. 21. MCWILLIAM, P., FARRAR, G. J., KENNA, P., BRADLEY, D. G., HUMPHRIES, M. M., SHARP, E. M., MCCONNELL, D. J., LAWLER, M., SHEILS, D., RYAN, C., STEVENS, K., DAIGER, S. P., AND HUMPHRIES, P. (1989). Autosomal dominant retinitis pigmentosa (ADRP): Localization of an ADRP gene to the long arm of chromosome 3. Genomics 6: 619-622. 22. NELSON, M., AND MCCLELLAND, M. (1987). The effect of sitespecific methylation on restriction-modification enzymes. Nucleic Acids Res. (Supplement) 15: 219-230. 23. OTT, J. (1974). Estimation of the recombination fraction in human pedigrees: Efficient computation of the likelihood for human linkage studies. Amer. J. Hum. Genet. 26: 588-597. 24. SEALEY, P. G., WHI?TAKER, P. A., AND SOUTHERN,E. M. (1985).
IN
AUTOSOMAL
DOMINANT
RP
173
Removal of repeat sequences from hybridisation probes. Nucleic Res. 13: 1905-1922. 25. SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517. 26. SPENCE, M. A., SPARKES, R. S., HECKENLIVELY, J. R., PEARLMAN, J. T., ZEDALIS, D., SPARKES, M., GRIST, M., AND TIDEMAN, S. (1977). Probable genetic linkage between autosomaI dominant retinitis pigmetosa (RP) and amylase (AMYB): Evidence of an RP locus on chromosome 1. Amer. J. Hum. Genet. 27: 397404; Erratum. Amer. J. Hum. Genet. 29: 592. 27. YIJIAN, F., QIANXUN, F., AND CHENGREN, L. (1987). Linkage analysis of autosomal dominant retinitis pigmentosa (RP) in China: Evidence of an RP locus on chromosome 1: Ninth International Workshop on Human Gene Mapping. Cytogenet. Cell Genet. 46: 614. Acids
6.168-173
(1990)
No Evidence for Linkage between Late Onset Autosomal Dominant Retinitis Pigmentosa and Chromosome 3 Locus D3S47 (Cl 7): Evidence for Genetic Heterogeneity C. F. INGLEHEARN,* “Molecular
M. JAy,t D. H. LESTER,* R. BASHIR,* B. JAY,t A. C. BIRD,t A. F. WRIGHT,* H. J. EVANS,* S. S. PAPIHA,* AND 5. S. BHATTACHARYA**’
Genetics Unit, Human Genetics, University of Newcastle upon Tyne; tDepartment of Clinical Ophthalmology, Moorfields Eye Hospital, City Road, London; and $MRC Human Genetics Unit, Western General Hospital, Edinburgh, United Kingdom Received
September
18, 1989;
INTRODUCTION
Retinitis pigmentosa (RP) is one of the most common human inherited eye disorders and accounts for approximately 7% of blindness in the United Kingdom (DHSS, 1979). Typically, patients complain of night blindness and progressive narrowing of the visual field. Clinically it is characterized in later stages by bonespiculelike pigmentation in the midperiphery of the retina and a severely attenuated or nonrecordable rod electroretinogram (reviewed by Krill, 1972). RP is familial and occurs in X-linked, autosomal recessive, and autosomal dominant forms. The autosomal dominant category represents approximately 25% of total RP in the British population (Bundey and Crews, 1984). There is substantial variation in prognosis within this grouping, suggesting an underlying genetic heterogeneity. Berson et al. (1969) suggestedthat families could 1 TO whom correspondence should be addressed at University of Newcastle upon Tyne, Molecular Genetics Unit, Upper Claremont Street, Newcastle upon Tyne NE2 4AJ, UK.
$3.ocl
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
October
24, 1989
be classified on the basis of the degree of gene penetrance. A further classification put forward by Massof and Finkelstein (1979) subdivided ADRP on the basis of differential effects on rod relative to cone sensitivity of long- and short-wavelength stimuli. The same authors also suggested a division based on age of onset: type I (early onset) with night blindness before 10 years, and type II (late onset) beginning in the third decade (Massof and Finkelstein, 1981). More recently ADRP has been subdivided on the basis of persistence of a measurable rod electroretinogram (Arden et al., 1983) and also on distribution of pigmentation in an affected retina in the early stages of the disease (Lyness et al., 1985; Farber et al., 1985). A classification on the latter basis gives the diffuse (D) and regional (R) types, which correspond to the early (type I) and late (type II) onset categories of Massof and Finkelstein (1981), respectively. Variation in the disease status within these groups and even within families makes it impossible, however, to determine whether these clinical categories correspond to different genetic lesions. Use of a linked genetic marker would make it possible to determine whether these differences are allelic or result from defects in different genes. Markers that are closely linked to the ADRP phenotype within families would also be valuable tools to genetic counselors, allowing more accurate detection of gene carriers and of disease status in prenatal diagnoses. Ultimately, close genetic linkage may also lead to isolation of the defective gene itself, which would increase an understanding of the molecular pathology of this complex disease and may facilitate therapeutic intervention at a presymptomatic stage. The first recorded linkage study in ADRP examined four extended pedigrees using a number of well-defined
Retinitis pigmentosa is an inherited form of blindness caused by progressive retinal degeneration. P. McWilliam et ul. (1989, Genomics 5: 619-622) demonstrated close genetic linkage between autosomal dominant retinitis pigmentosa (ADRP) and locus D3S47 (C17) in a single early onset pedigree. The marker Cl7 maps to the long arm of chromosome 3. Clinically, the disease phenotype has been subdivided into at least two forms on the basis of age of onset, as well as electrodiagnostic criteria. We demonstrate that Cl7 is unlinked in a late onset pedigree, indicating that the phenotypic variation seen reflects underlying genetic heterogeneity. 0 1~90 Academic Pre~e, Inc.
os&u3-7543/90
revised
168
LOCUS
HETEROGENEITY
IN
biochemical markers and pooling the results of the families in calculating lod scores (Hussels-Maumenee et al., 1975). No significant linkage was observed. Such an approach, however, would be flawed if genetic heterogeneity existed. A similar study, using only a single large pedigree (Spence et al., 1977), reported a maximum lod score of 2.5 at a recombination fraction of 0.10 to rhesus on chromosome 1. However, further data on the same family decreased that figure to 2.13 (Heckenlively et al., 1982). A study that pooled data from that pedigree and four others obtained a lod score of 1.51 between rhesus and ADRP, while all other biochemical markers tested gave significantly smaller lod scores (Field et aZ., 1982). Pooling of data from a further two families gave a final lod score of 1.89 to rhesus, still not reaching the generally accepted required value of 3 for statistical significance (Daiger et al., 1987). Results from a Chinese study further support a chromosome 1 linkage but remain unconfirmed (Yijian et al., 1987), while studies using DNA probes around rhesus have found no linkage in two large pedigrees (Bradley et al., 1989; Inglehearn et al., 1989). Recently, as a result of extensive linkage studies using the probes reported by Donis-Keller et al. (1987), McWilliam et al. (1989) have reported tight linkage between ADRP and a cosmid clone, C17, mapping to chromosome 3q in a single large type I (early onset) Irish pedigree. In this paper, we present results of a genetic linkage study between Cl7 and a large R type (type II) late onset pedigree with partial penetrance. Cl7 was found to be unlinked in this family, which confirms the existence of genetic heterogeneity within ADRP as suggested by clinical observations. MATERIALS
AND
METHODS
The approach of Don&-Keller et al. (1987) in constructing a complete linkage map of the human genome favored the use of whole cosmid and phage clones, since, when adequately competed, a large insert is more likely to detect a polymorphism. Probe Cl7 is a 40-kb insert isolated from an Mb01 partial digest library of the human genome constructed in cosmid vector c2RB (Collaborative Research Inc.). The cosmid clone was grown by alkaline lysis maxiprep, as described by Maniatis et al. (1982). Human genomic DNAs were prepared from lymphocytes obtained from lo-ml blood samples by the method described by Herman and Frischauf (1987). Restriction digestion was carried out in buffer supplied by the manufacturer (NBL), for between 4 and 16 h, to between 2 and 20 times the recommended required digestion. TaqI digests were carried out at 65”C, while MspI digests appeared to work best at room temperatures (20-25 “C) as recommended by Collaborative Research Inc. Samples were size-fractionated on
AUTOSOMAL
DOMINANT
RP
169
1% agarose gels run overnight at 1.5 to 2 V/cm. Gels were denatured at high pH, neutralized, and then blotted to Hybond-N nylon membranes (Amersham) by the method of Southern (1975). DNA was fixed to the membranes by uv transillumination and then air-dried for 24 h. Filters were prehybridized in 0.5 M sodium phosphate buffer and 7% SDS for approximately 30 min and then hybridized in the same solution but with 10% dextran sulfate and radiolabeled probe. Whole cosmid Cl7 (50 ng) was oligo-labeled with [32P]dTTP by the method of Feinberg and Vogelstein (1983) and purified on a Sephadex G-50 column (Pharmacia) to remove unincorporated nucleotides. The labeled cosmid was then competitively hybridized to sheared total human DNA to reduce lane background caused by common repeats in the cosmid (Sealey et aZ., 1985). Labeled probe (50 ng) was competed against 4 mg total human DNA in 5X SSC in a total volume of 1.3 ml at 65°C for 30 min. The mix was then added to 20 ml of the hybridization solution. Filters were hybridized in polythene bags overnight at 65”C, rinsed in 2X SSC and 0.1% SDS at room temperature, and washed twice at 65°C in 0.5X SSC and 0.1% SDS. Autoradiographic images were obtained by exposure for between 1 and 4 days at -80°C to Kodak XAR5 film in Kodak regular cassettes. Two-point lod scores were calculated using the computer program LIPED (Ott, 1974). RESULTS
Polymorphisms Detected by Cl 7 Figure 1 shows panels of DNA from five unrelated individuals. The DNAs were digested with enzymes TaqI and MspI and then probed with competed C17. Twelve bands can be seen on an MspI digest. The top two bands, at 14 and 12.3 kb, are allelic. Frequencies of the upper (A) allele and lower (a) allele in 44 unrelated individuals were 0.35 and 0.65, respectively. Frequencies within family ADRP5 were 0.36 for A and 0.64 for a. This has proved to be the most informative allele system detected by this probe in ADRP5. Polymorphic bands can also be seen at approximately 8.7 and 6.7 kb, while weaker bands appear at approximately 5.5 and 3.5 kb. However, in some individuals, bands at 6.7 and 8.7 kb were either much weaker or totally absent, while the lower bands were correspondingly stronger (data not shown). The enzyme MspI recognizes the motif CCGG and cuts regardless of whether the second C is methylated. However, if this sequence is preceded by two guanine residues, to give GGCCGG, then the enzyme will cut only an unmethylated site (Nelson and McClelland, 1987). It is probable therefore that this second allele system is bounded on one side by such a site. In reactions sampled at time points of
170
INGLEHEARN
ET
Normal DNAs
could also be seen with this probe at approximately 3.2, 2.4, 2.3, 1.9, 1.2, 1.0, and 0.75 kb. In TuqI digests the Cl7 probe detects 13 bands. Again the upper two bands, at approximately 6.8 and 7.2 kb, are allelic. In 45 unrelated individuals the upper allele (T) was found to have a frequency of 0.17 and the lower (t) 0.83, while in the ADRP5 family, frequencies are 0.23 and 0.77, respectively. In samples that are visibly discernible as partial digests, an extra band can be seen at 6.3 kb. However, the band at 4.4 kb is clearly not the result of partial digestion and appears to represent one allele of a second polymorphism detected with this probe on Tag1 digests. Since the second allele could not be interpreted with certainty, this polymorphic system was not used in linkage analysis of ADRP. Constant bands in TuqI digests were seen at sizes of approximately 6.5, 4.2, 4.0, 3.2, 2.6, 2.3, 1.9, 1.7, 1.0, and 0.6 kb. Clinical
Cl7
increasing digestion, the lower bands were found to increase in strength but then stop at a point that varied in different individuals, suggesting differing degrees of methylation at this site (data not shown). Upper bands never disappeared completely in individuals tested in this way, although these bands had been seen to do so in other individuals. Because of the potential ambiguity in interpretation of this polymorphism, a lod score was not calculated for this system. Constant MspI bands
BP tt
2
l n 2.
Pedigree
Affected
ADRPB
0
showing
0
Unaffected
autosomal
Classification
of the Family
Studied
Figure 2 shows the complete pedigree known as ADRP5. It can be clearly seen that the pattern of inheritance in this family is autosomal dominant. By carrying out linkage studies only in a single large pedigree, we remove any problem associated with heterogeneity of disease locus in ADRP. Shaded symbols represent individuals affected with retinitis pigmentosa. However, the extent to which individuals are affected varies greatly. Onset of the defect may be at any time between 15 and 40 years of age, with night blindness as the first symptom. In some individuals the defect has led to complete loss of sight, although others retain good visual acuity until relatively late in life. In three
FIG. 1. DNA, MspI and TagI digested and probed with competed C17, from six unrelated individuals. Allelic bands used in the linkage analysis are indicated by arrowheads.
FIG.
AL.
@
dominant
q
::
saMk Tf
Unaffected below 40years
segregation
of the type
II late onset phenotype.
Tt
tt
Aa
Tt
Aa It
LOCUS
HETEROGENEITY
IN
AUTOSOMAL
DOMINANT
171
RP
ADRPS
1
2
3
4
5
6
7
FIG. 3. Genomic DNAs from ADRP5, MspI (upper) to individuals numbered on the pedigree above. Several yet16is homozygous AA and 20ishomozygous aa.
8
9 IO 11 12 13 14 15 16 17 18 19 20 21
and ToqI (lower) digested and probed with competed C17. Track numbers correspond crossovers can be seen; for example, tracks 16 and 20 have DNAs from affected sibs
cases, individuals with affected offspring, who were therefore obligate carriers, were so mildly affected that they were unaware of any problems. The only detectable defect in these patients was a slight elevation of rod thresholds on static perimetry testing as described by Ernst et al. (1983). To counter the problem of partial penetrance, we have used only data from affected individuals and from those above the age of 40, who were still unaffected. The pattern of pigmentation in the retinas of affected individuals and age of onset in this family are consistent with regional (R type) RP. Linkuge Analysis Genomic DNAs from the pedigree shown were digested with restriction enzymes TaqI and MspI, re-
TABLE Lod Scores
between
spectively, blotted, and probed with C17. Alleles for the upper MspI and upper TaqI systems for the entire family are shown in Fig. 2. Autoradiographs for part of the family highlighting a number of crossovers are shown in Fig. 3. Table 1 shows the results of two-point linkage analysis between ADRP and the two polymorphisms in this family. The number of informative meioses for the MspI polymorphism was larger than that for the TaqI polymorphism and this can be seen from the distribution of alleles at each of the two sites, as shown in Fig. 2. However, negative lod scores for both polymorphisms are still seen up to a recombination fraction of 0.30. Therefore, it is clear that both polymorphic sites identified by the probe Cl7 are unlinked to the ADRP phenotype in this family.
1
C17-Associated Restriction Fragment Length Polymorphisms Dominant Retinitis Pigmentosa Family Recombination
fraction
and an Autosomal
(0)
Main locus
Enzyme
0.000
0.001
0.05
0.10
0.20
0.30
ADRP ADRP
MspI Td
-
-12.593 -5.865
-3.917 -2.298
-2.265 -1.586
-0.772 -0.781
-0.178 -0.312
0.40
0.50
0.009 -0.086
0.000 0.000
172
INGLEHEARN DISCUSSION
The results presented here, considered together with those of McWilliam et al. (1989), prove that there is genetic heterogeneity in autosomal dominant retinitis pigmentosa. Further experiments underway in our laboratory should soon determine whether the Cl7 linkage demonstrated by McWilliam and co-workers holds true for all early onset (diffuse) ADRP families or whether there is further genetic variation within this group. In addition, in all the families that prove to be Cl7 unlinked, the task of searching for linkage will continue. It is interesting to note that Field et al. (1982) presented data that excluded an ADRP gene from nearly 40 CM around the transferrin (TF) locus, at 3q21. As yet, probe Cl7 has only been mapped relative to other probes from Collaborative Research and may therefore be some distance from TF, although their chromosomal locations appear to correspond. Alternatively, the exclusion of an ADRP locus from the region containing TF by Field and co-workers may have been due to the pooling of data from what we now know to be C17linked and unlinked families. Transferrin is a highly polymorphic genetic system and it will be of interest to analyze further the linkage relationships in C17linked ADRP families. In the short term, heterogeneity within ADRP families will limit the usefulness of the reported linkage in counseling and carrier diagnosis to those families that demonstrate close linkage with C17. The information on the use of the polymorphisms detected by Cl7 presented here should be of value to other researchers studying ADRP families, since the use of several polymorphic systems should make the probe informative in most pedigrees. ACKNOWLEDGMENTS We are grateful to the American Retinitis Pigmentosa Foundation and the George Gund Foundation for their generous support of this work. We thank Helen Ackford, Dawn Thistleton, and Alison Hardcastle for excellent technical support. Thanks also to Dr. Peter Humphries for informing us of his results before publication. Our special thanks to Dr. Anthony Moore for clinical assessment of members of this ADRP family. Finally, we are grateful to Pauline Battista for her patience in typing this manuscript. REFERENCES 1. ARDEN, G. B., CARTER, R. M., HOGG, C. R., POWELL, D. J., ERNST, W. J. K., CLOVER, G. M., LYNESS, A, L., AND QIJINLAN, M. P. (1983). Rod and cone activity in patients with dominantly inherited retinitis pigmentosa: Comparisons between psychophysical and electroretinographic measurements. &it. J. Ophthalmol. 67: 405-418. 2. BERSON, E. L., GOURAS, P., GUNKEL, R. D., AND MYRIANTHOPOULOS, N. C. (1969). Dominant retinitis pigmentosa with reduced penetrance. Arch. Ophthalmol. 99: 226-234.
ET AL. 3. BRADLEY, D. G., FARRAR, G. J., SHARP, E. M., KENNA, P., HUMPHRIES, M. M., MCCONNELL, D. J., DAIGER, S. P., MCWILLIAM, P., AND HUMPHRIES, P. (1989). Autosomal dominant retinitis pigmentosa: Exclusion of the gene from the short arm of chromosome 1 including the region surrounding the rhesus locus. Amer.
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13. HERMAN, B. G., AND FRISCHAUF,A. (1987). In “Guide to Molecular Cloning Techniques” (S. L. Berger and A. R. Kimmel, Eds.), pp, 180-183, Academic Press, London. 14. HUSSELS-MAUMENEE, I., PIERCE, E. R., BIAS, W. B., AND SCHLEUTERMANN, D. A. (1975). Linkage studies of typical retinitis pigmentosa and common markers. Amer. J. Hum. Genet. 27: 505-508. 15. INGLEHEARN, C. F., PAPIHA, S. S., JAY, M., MOORE, T., AND BHATTACHARYA, S. S. (1989). Linkage of internal minisatellite loci on chromosome 1 and exclusion of autosomal dominant retinitis pigmentosa proximal to rhesus. J. Med. Genet., in press. 16. KRILL, A. E. (1972). Retinitis pigmentosa: A review. Sight Sav. Rev. 42: 21-28. 17. LYNESS, A. L., ERNST, W., QUINLAN, M. P., GLOVER, G. M., ARDEN, G. B., CARTER, R. M., BIRD, A. C., AND PARKER, J. A. (1985). A clinical, psychophysical, and electroretinographic survey of patients with autosomal dominant retinitis pigmentosa. Brit. J. Ophthalmol. 69: 326-339. 18. MANIATIS, T., FRITSCH, E. F., AND SAMBROOK, J. (1982). “MOlecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 19. MASSOF, R. W., AND FINKELSTEIN, D. (1979). Rod sensitivity relative to cone sensitivity in retinitis pigmentosa. Invest. Ophthalmol. Visual Sci. 18: 263-272.
LOCUS
HETEROGENEITY
20. MASSOF, R. W., AND FINKELSTEIN, D. (1981). Two forms of autosomal dominant retinitis pigmentosa. Dot. Ophthuhnol. 61: 289-346. 21. MCWILLIAM, P., FARRAR, G. J., KENNA, P., BRADLEY, D. G., HUMPHRIES, M. M., SHARP, E. M., MCCONNELL, D. J., LAWLER, M., SHEILS, D., RYAN, C., STEVENS, K., DAIGER, S. P., AND HUMPHRIES, P. (1989). Autosomal dominant retinitis pigmentosa (ADRP): Localization of an ADRP gene to the long arm of chromosome 3. Genomics 6: 619-622. 22. NELSON, M., AND MCCLELLAND, M. (1987). The effect of sitespecific methylation on restriction-modification enzymes. Nucleic Acids Res. (Supplement) 15: 219-230. 23. OTT, J. (1974). Estimation of the recombination fraction in human pedigrees: Efficient computation of the likelihood for human linkage studies. Amer. J. Hum. Genet. 26: 588-597. 24. SEALEY, P. G., WHI?TAKER, P. A., AND SOUTHERN,E. M. (1985).
IN
AUTOSOMAL
DOMINANT
RP
173
Removal of repeat sequences from hybridisation probes. Nucleic Res. 13: 1905-1922. 25. SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517. 26. SPENCE, M. A., SPARKES, R. S., HECKENLIVELY, J. R., PEARLMAN, J. T., ZEDALIS, D., SPARKES, M., GRIST, M., AND TIDEMAN, S. (1977). Probable genetic linkage between autosomaI dominant retinitis pigmetosa (RP) and amylase (AMYB): Evidence of an RP locus on chromosome 1. Amer. J. Hum. Genet. 27: 397404; Erratum. Amer. J. Hum. Genet. 29: 592. 27. YIJIAN, F., QIANXUN, F., AND CHENGREN, L. (1987). Linkage analysis of autosomal dominant retinitis pigmentosa (RP) in China: Evidence of an RP locus on chromosome 1: Ninth International Workshop on Human Gene Mapping. Cytogenet. Cell Genet. 46: 614. Acids
