Proc. Natl. Acad. Sci. USA Vol. 88, pp. 6481-6485, August 1991 Genetics Rhodopsin mutations in autosomal dominant pigmentosa CHING-HWA SUNG*, CAROL M. DAVENPORT*, JILL C. HENNESSEYt, IRENE H. MAUMENEE*, SAMUEL G. JACOBSON§, JOHN R. HECKENLIVELY¶, RODNEY NOWAKOWSKI II, GERALD FISHMAN**, PETER GOURAStt, AND JEREMY NATHANS*#* *Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, and Department of Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205; tNational Foundation, 1401 Mount Royal Avenue, Baltimore, MD 21217; tWilmer Ophthalmologic Institute, Johns Hopkins University School of Medicine, 600 E. Monument Street, Baltimore, MD 21205; §Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, FL 33101; 1Jules Stein Eye Institute, University of California at Los Angeles School of Medicine, Los Angeles, CA 90024; 1School of Optometry, University of Alabama at Birmingham, Birmingham, AL 35294; **Department of , University of Illinois College of Medicine, Chicago, IL 60612; and ttDepartment of Ophthalmology, Columbia University College of Physicians and Surgeons, New York, NY 10032 Communicated by Victor A. McKusick, April 22, 1991

ABSTRACT DNA samples from 161 unrelated patients given candidate gene is, therefore, unlikely to be present in with autosomal dominant retinitis pigmentosa were screened more than a fraction of the retinitis pigmentosa population. for point mutations in the rhodopsin gene by using the poly- To search efficiently for rhodopsin mutations, our strategy merase chain reaction and denaturing gradient gel electropho- has been to collect DNA samples from a large number of resis. Thirty-nine patients were found to carry 1 of 13 different unrelated patients with retinitis pigmentosa, amplify the point mutations at 12 amino acid positions. The presence or rhodopsin gene exons by using PCR (9), and then assay the absence of the mutations correlated with the presence or amplified products for sequence variation by using denatur- absence of retinitis pigmentosa in 174 out of 179 individuals ing gradient gel electrophoresis (DGGE) (10). We focused tested in 17 families. The mutations were absent from 118 initially on 161 unrelated patients with autosomal dominant control subjects with normal vision. retinitis pigmentosa (ADRP). This choice was prompted by the report of McWilliam et al. (11) that in one large Irish vision is mediated by four visual pigments. family the gene responsible for ADRP cosegregates with markers tightly linked to the rhodopsin gene on the long arm Rhodopsin, the pigment in rods, mediates vision in dim light; of chromosome 3 (2). the red-, green-, and blue-sensitive pigments reside in the Here we report that 39 out of 161 unrelated patients with cones and mediate . The visual pigments form a ADRP carry point mutations in the coding sequence of the family ofhomologous proteins encoded by the corresponding rhodopsin gene. The mutations produce 13 predicted amino members of a family of genes (1, 2). acid substitutions at 12 different positions in the rhodopsin The goal ofthe present study is to identify mutations in the molecule. Related results have been reported by Dryja and gene encoding human rhodopsin. In planning this study, we Bhattacharya and their colleagues (12-14) in screening for first considered the phenotypes that result from alterations in rhodopsin gene mutations in ADRP patients. the genes encoding the human cone pigments. One class of rearrangements in the red and green pigment gene cluster preserves the structural integrity ofthe encoded proteins and MATERIALS AND METHODS causes the benign anomalies of color vision commonly re- Sample Collection and Processing. Participants were re- ferred to as (3). A second class of mutations cruited by their ophthalmologists- (I.H.M., S.G.J., G.F., is responsible for , a dysfunction of J.R.H., and P.G.), optometrist (R.N.), or through the com- both red and green cones that is associated in some individ- puterized Retinitis Pigmentosa Foundation National Registry uals with a progressive degeneration ofthe cone-rich central (J.C.H.). Control samples were obtained from students at the (4, 5); and a third type of mutation causes red cone United States Air Force Academy. DNA was prepared from dysfunction and a progressive central degeneration (6). Re- venous blood by proteinase K digestion, followed by equilib- cent experiments show that point mutations in the blue rium centrifugation in a cesium chloride density gradient (1). pigment gene lead to defective blue cone function (C. Weitz PCR Amplification and-DGGE. Seven segments of the and J.N., unpublished results). rhodopsin gene encompassingtheentire coding region (1) were By analogy with the known cone pigment gene defects, we amplified by PCR using the following primer pairs [the nota- reasoned that rhodopsin gene mutations would produce a tion (GC) indicates the sequence CGCCCGCCGCGC- deficit in rod photoreceptor function (i.e., night blindness) CCCGCGCCCGCCCCGCCGCCCCCGCCC added to the 5' and might lead to a progressive degeneration and consequent end of the oligonucleotide (10)]: exon 1 5' half (1L), TTCG- loss of function in the rod-rich peripheral retina. Night CAGCATTCTTGGGTGG and (GC)TGTGCTGGACGGT- blindness and progressive loss of peripheral vision are the GACGTAGAGCGTGAGGAA; exon 1 3' half (1R), ATGC- hallmarks of retinitis pigmentosa, a group of inherited disor- TGGCCGCCTACATGYT and (GC)AAGCCCGGGACTCT- ders that appear to affect the photoreceptors and underlying CCCAGACCCCTCCATGC;exon2,TGCACCCTCCTTAGG- pigment epithelium (7). Retinitis pigmentosa is known to be CAGTG and (GC)AGACACTACTGGG'TlGAGTCCTGA- genetically heterogeneous, comprising X chromosome linked, autosomal recessive, and autosomal dominant types Abbreviations: ADRP, autosomal dominant retinitis pigmentosa; (8). Within each genetic type there is marked individual DGGE, denaturing gradient gel electrophoresis; amino acid substi- variation in natural history. Any particular mutation in a tutions are referred to by the single-letter amino acid designation of the wild-type residue, followed by its position number in the poly- peptide chain and the single-letter amino acid designation of the The publication costs of this article were defrayed in part by page charge mutant residue (e.g., P23H refers to the replacement of proline-23 by payment. This article must therefore be hereby marked "advertisement" histidine). in accordance with 18 U.S.C. §1734 solely to indicate this fact. #To whom reprint requests should be addressed.

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M WT P347L A C G T A C GIT FIG. 2. DNA sequences of wild-type and mutant PCR products. For each ofthe 13 different ADRP mutations, one example is shown ofwild-type and mutant DNA sequences in the neighborhood of the mutation. The C - I. mutations are cytidine 344-* thymidine (C344T) (A), C362A (B), C427T (C), T C467G (D), T554A (E), G560A (F), G610T (G), G2481T and G2482T (H), C.------C C2480T (I), A3815G (J), A3851G (K), C5261T (L), C5271 T (M). As an adjunct to the pedigree analysis, we compared the families, it will now be possible to provide genetic counseling frequency of each mutant allele in the ADRP population (n = and prenatal diagnosis based on DNA analysis. 161) and in a-control population of young adults with normal It is possible that some mutations escaped detection by the vision (n = 118). One mutant allele, P23H, is present in 24 of PCR/DGGE screening method employed in this study. Intron 161 unrelated ADRP subjects and is absent from the 118 and flanking region mutations outside of the amplified seg- control subjects. The other 12 mutations were found either ments would not be detected. Deletions or other rearrange- once or twice in the 161 ADRP subjects; they were not seen ments encompassing one ofthe primer sites would also escape in the control populationC~~~~~~~(Table 1). Altogether, rhodopsin detection because a normal PCR product would be produced mutations were observed in 39 of 161 ADRP subjects. from the wild-type chromosome. However, it is unlikely that point mutations residing within the amplified regions would go undetected by DGGE. Sheffield et al. (10) showed that >90%o DISCUSSION of single base changes produce a PCR product of variant Identification of Rhodopsin Mutations. The results pre- mobility when amplification is performed with a GC clamp sented here demonstrate that 24% of 161 unrelated ADRP primer. Consistent with that observation, only 1 of 18 variant patients carry point mutations in the gene encoding bands observed in the present study comigrated with the wild rhodopsin. As the sample population in this study was almost type under the conditions employed (HS698; exon 1R; see Fig. exclusively Caucasian, this value may differ in other popu- 1). Because each subject is likely to be heterozygous for the lation groups. The presence or absence of rhodopsin muta- dominant mutation, heteroduplexes carrying a base mismatch tions correlates with the presence or absence ofADRP in 174 are expected to form during the annealing step ofthe last PCR of 179 cycle. In each of the 18 variant patterns, the heteroduplexes family members tested (including probands). The four show a prominent decrease in mobility compared to the individuals in one family who carry mutations but have no corresponding homoduplexes. visual symptoms may represent examples of incomplete Heterogeneity of Rhodopsin Mutations. In this study 13 penetrance, delayed onset ofthe disease, or very mild disease different mutations were observed at 12 amino acid positions expression. For several alleles the pedigrees are too small for within the rhodopsin coding region. This allelic heterogeneity cosegregation to be statistically significant. Analysis of ad- suggests a corresponding variability in biochemical defects ditional families or identification of a biochemical defect in and in the natural history of the associated retinal disease. the mutated rhodopsins is needed to confirm a role for these Factors other than heterogeneity at the rhodopsin locus may alleles in producing retinitis pigmentosa. The single most also contribute to phenotypic variability. Indeed, two recent common allele, P23H, was found in 15% of the ADRP studies have found marked variation in natural history among families. These data are in agreement with those of Dryja et patients with the P23H mutation (15, 16). al. (12) who found the P23H mutation in 17 of 148 unrelated Additional rhodopsin gene mutations are likely to exist in ADRP subjects. In the present study no other allele was the human gene pool. They may account for the retinal found in more than two families. For one-quarter of ADRP degeneration or functional impairment in such disorders as Downloaded by guest on September 26, 2021 6484 Genetics: Sung et al. Proc. Natl. Acad. Sci. USA 88 (1991) A Bi B2

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FIG. 3. Pedigrees of ADRP families. A history of night blindness and/or loss were considered diagnostic of retinitis pigmentosa. Most of the affected participants have been independently evaluated by an ophthalmologist. A question mark indicates an uncertain diagnosis. Numbers indicate the family members tested; arrows indicate probands. Families designated by the same letter carry the same mutation (e.g., BI and B2). Subject 11 in family D and subjects 5, 6, and 7 in family L are classified as affected based upon measurements of rod electroretinograms, dark adaptometry, and rhodopsin levels; they report no . autosomal recessive retinitis pigmentosa, Leber's congenital Naturally occurring variants have been extremely useful in amaurosis, or stationary night blindness. It will also be studies of protein structure and function. The variant interesting to determine that fraction of simplex retinitis rhodopsins responsible for ADRP could be defective in pigmentosa patients who carry new rhodopsin mutations. protein folding/stability, intracellular targeting, signal trans- The present study indicates that neutral amino acid sub- duction, or as-yet-undefined functions. The availability of stitutions in rhodopsin are extremely rare. Of the 322 tissue culture expression systems for rhodopsin will permit rhodopsin genes screened, 39 carry mutations responsible for the production of each variant for detailed study (18, 19). ADRP and none carry aht amino acid substitution unrelated to Implications for Large-Scale Genetic Screening of Hetero. ADRP. This finding rules out rhodopsin gene polymorphism geneous Disorders. For many inherited disorders, clinical as a source of the reported common variation in the point of variability suggests a corresponding heterogeneity in under- maximal sensitivity of human rod vision (17). lying genetic defects. One approach to identifying the causal Downloaded by guest on September 26, 2021 Genetics: Sung et al. Proc. Natl. Acad. Sci. USA 88 (1991) 6485

A B2 E F2 H2 I1 J *1 _ _ * *1 * * 2 2 * 2 2 2 *2 2 3 * 3 - 3 * 3 * 3 3 3 _ 4 *4 4 4 * 4 * 4 * 5- * 5 5 *5 5 *6 _ 6 5 6 6 Fl * 6 *7 _ * 7 - 7 * 7 8 8 G *9- * 10 * 2 I2 9 B1 * 3 - * 10- 10 *4 --2 12 * 2 _ C 5 *3 _ *13 * 2 - *6 3 K 3 *4_ *14 * 4 - * _ 5 Iso * 4 - *2- 8 15 * 5 * 2 5 *3 - *9 6 _ *16 * 6 - 3 * 6 - * 7 - 4 *10 - 8*_ 17 4 7 * 8 - * 5 8 - *5 - Ill * 6 12 *19 9 6 9 *7 - *13 Hi 20 * 0 - 7 * 10 - 8 *14 *21 * 11 _ *1 - *22_ * 12 - 15 *2 *23 - L 13 *3 _24 _4 * 14 - - 25 * I- 18 * 15 - *1 26 * 2 19 *6 - - * 16 - *2 *27 * 3 -3 *20 * 7 ' _ *28 - 4 FIG. 4. Segregation of rho- 4 21 *29 _ 5 *22 * 5- dopsin mutations in ADRP families 30 * 6 *6 *23 *31 _ * 7 by hybridization of mutant allele- 7 24 *32 specific 13-mers to PCR products 8 25 33 *9--- 26 from family members indicated in *10 - 27 Fig. 3. Asterisks denote individu- * 11 als with retinitis pigmentosa. mutations is to choose as candidates those genes whose participating; Drs. Charles Weitz, Mark Gray, Monica Traystman, products are active in the relevant physiological processes. and Haig Kazazian for DGGE advice; Dr. Clark Riley, Ms. Anatoli To overcome the problem of genetic heterogeneity, a large Collector, Ms. Cynthia Wendling, and Ms. Jodie Franklin for syn- number of unrelated can be screened for mutations thetic oligonucleotides; Drs. David Valle, Daniel Nathans, Donald patients Zack, and Charles Weitz for helpful comments on the manuscript; in the candidate genes. As demonstrated in this study, the and Ms. Teri Chase for expert secretarial assistance. This work was combination ofPCR and DGGE is well suited for the efficient supported by the Howard Hughes Medical Institute and the National detection ofpoint mutations in a large patient population. The Retinitis Pigmentosa Foundation. efficiency of these methods makes feasible the screening of disorders for which a genetic component is known but for 1. Nathans, J. & Hogness, D. S. (1984) Proc. Nati. Acad. Sci. USA 81, not 4851-4855. which simple Mendelian inheritance has been demon- 2. Nathans, J., Thomas, D. & Hogness, D. S. (1986) Science 232, 193-202. strated. 3. Nathans, J., Piantanida, T. P., Eddy, R. L., Shows, T. B. & Hogness, D. S. (1986) Science 232, 203-210. We thank all of the ADRP subjects and their relatives for partic- 4. Fleischman, J. A. & O'Donnell, F. E. (1981) Arch. Ophthalmol. 99, ipating; Ms. Rosalind Palmer and her at the Wilmer 468-472. colleagues 5. Nathans, J., Davenport, C. M., Maumenee, 1. H., Lewis, R. A., Hejt- Retinitis Pigmentosa Center for assistance in identifying patients and mancik, F., Litt, M., Lovrien, E., Weleber, R., Bachynski, B., Zwas, F., collecting blood samples; Ms. Pat Catlin, Drs. John Jernigan, and Klingaman, R. & Fishman, G. (1989) Science 245, 831-838. Thomas Loftus and the cadets of the U.S. Air Force Academy for 6. Reichel, E., Bruce, A. M., Sandberg, M. A. & Berson, E. L. (1989) Am. J. Ophthalmol. 108, 540-547. Table 1. Frequency of rhodopsin mutations in ADRP and 7. Heckenlively, J. R. (1988) Retinitis Pigmentosa (Lippincott, Philadel- control populations phia). 8. Boughman, J. A. & Fishman, G. A. (1983) Br. J. Ophthalmol. 67, 449-454. Mutation ADRP Control 9. Mullis, K. B. & Faloona, F. A. (1987) Methods Enzymol. 155, 335-350. 10. Sheffield, V. C., Cox, D. R., Lerman, L. S. & Myers, R. M. (1989) Proc. T17M 1 0 Nati. Acad. Sci. USA 86, 232-236. P23H 24 0 11. McWilliam, P., Farrar, G. J., Kenna, P., Bradley, D. G., Humphries, M. M., Sharp, E. M., McConnell, D. J., Lawler, M., Sheils, D., Ryan, F45L 1 0 C., Stevens, K., Daiger, S. P. & Humphries, P. (1989) Genomics 5, T58R 1 0 619-622. V87D 1 0 12. Dryja, T. P., McGee, T. L., Reichel, E., Hahn, L. B., Cowley, G. S., G89D 2 0 Yandell, D. W., Sandberg, M. A. & Berson, E. L. (1990) Nature (Lon- don) 343, 364-366. G106W 1 0 13. Dryja, T. P., McGee, T. L., Hahn, L. B., Cowley, G. S., Olsson, J. E., R135L 2 0 Reichel, E., Sandberg, M. A. & Berson, E. L. (1990) N. Engl. J. Med. R135W 2 0 323, 1302-1307. Y178C 1 0 14. Inglehearn, C. F., Bashir, R., Lester, D. H., Jay, M., , A. C. & Bhattacharya, S. (1991) Am. J. Hum. Genet. 48, 26-30. D19OG 1 0 15. Berson, E. L., Rosner, B., Sandberg, M. A. & Dryja, T. P. (1991) Arch. Q344stop 1 0 Ophthalmol. 109, 92-101. P347L 1 0 16. Heckenlively, J. R., Rodriguez, J. A. & Daiger, S. P. (1991) Arch. Total 39 0 Ophthalmol. 109, 84-91. 17. Alpern, M. (1987) Vision Res. 27, 1471-1480. The frequency of each rhodopsin gene point mutation in 161 18. Oprian, D. D., Molday, R. S., Kaufman, R. J. & Khorana, H. G. (1987) unrelated subjects with ADRP and 118 subjects with normal vision Proc. Nati. Acad. Sci. USA 84, 8874-8878. is given. Mutations were detected as in Fig. 4. 19. Nathans, J., Weitz, C. J., Agarwal, N., Nir, I. & Papermaster, D. S. (1989) Vision Res. 29, 907-914. Downloaded by guest on September 26, 2021