Retinitis Punctata Albescens Associated With the Arg135Trp Mutation in the Rhodopsin Gene

ERIC SOUIED, MD., GISELE SOUBRANE, PH.D., PASCALE BENLIAN, MD., GABRIEL J. COSCAS, MD.,

SYLVIE GERBER, ARNOLD MUNNICH, PH.D., AND JOSSELINE KAPLAN, PH.D.

• PURPOSE: To screen for mutations in the rho­dopsin, peripherin/RDS, and ROM1 genes in a family affected with retinitis punctata albescens. Because clinical heterogeneity was observed in this family, with some members affected with retinitis punctata albescens and one member af­fected with features typical of retinitis pigmentosa, we analyzed the apolipoprotein E gene to elucidate this unusual intrafamilial heterogeneity. • METHODS: The coding sequences of these genes were analyzed with a combination of single-strand conformation polymorphism and direct sequence analysis. Haplotypes of the apolipoprotein E gene were analyzed by polymerase chain reaction and enzymatic digestion. • RESULTS: The Argl35Trp mutation in the rho­dopsin gene was observed in all affected members of this family, but no mutation was detected in the peripherin/RDS or ROM1 genes. The e4 allele of the apolipoprotein E gene apparently cosegregated with the albescens phenotype in this family. • CONCLUSIONS: The albescent phenotype in retinal dystrophy appears to not be caused exclu­sively by a peripherin/RDS gene mutation, and we

Accepted for publication July 10, 1995. From the Genetics Labotatory 1NSERM U-393, Hτpital des Enfants-

Malades, Paris (Drs. Souied, Gerber, Munnich, and Kaplan); Endocri­nology Department, Hτpital Pitiι-Salpιtriθre, Paris (Dr. Benlian); and Department of Ophthalmology, University Eye Clinic, Creteil (Drs. Souied, Soubrane, and Coscas), France. This study was supported by the Association Franηaise contre les Myopathies, Paris, France; the Association Franηaise Retinitis Pigmentosa, Paris, France; and the Fed­eration des Aveugles de France, Paris, France.

Reprint requests to Josseline Kaplan, Ph.D., Genetics Laboratory INSERM U-393, Hτpital des Enfants-Malades, 149, rue de Sθvres, 75743 Pans Cedex 15, France; fax: 331 44 49 51 50.

suggest that the apolipoprotein E gene may play a role in the albescent phenotype.

R ETINITIS PIGMENTOSA IS A HETEROGENEOUS

group of inherited degenerative retinal disor­ders characterized by a loss of photoreceptor

function.1 The incidence of retinitis pigmentosa is between one in 3,500 and one in 5,000,2'4 and it can be classified into autosomal dominant, autosomal recessive, X-linked, and sporadic types.5 Affected individuals typically display night blindness, progres­sive reduction of the visual field, and, in the more advanced stages, decreased visual acuity. Ocular findings include intraretinal pigment deposition, at­tenuated retinal vessels, waxy pallor of the optic disk, and abnormal electroretinographic response.6

We studied a French family affected with retinitis punctata albescens. Clinical heterogeneity was ob­served in this family; some members were affected with retinitis punctata albescens and one member displayed features typical of retinitis pigmentosa.

Eight genes for autosomal dominant retinitis pig­mentosa have been localized,7'14 and two of them, the rhodopsin and the peripherin/RDS genes, have been identified.1516 These two genes account for 30% of all cases of autosomal dominant retinitis pigmentosa.17

Rhodopsin is a photopigment involved in the initial steps of the visual transduction cascade and represents more than 80% of the constituent protein of the rod outer segment membrane; it is present at approximately 108 molecules per rod photoreceptor.18

The rhodopsin gene encompasses 7 kb on chromo­some 3q21 and contains five exons.15 More than 70 distinct rhodopsin gene mutations have been de-

VOL. 121, NO. 1 © AMERICAN JOURNAL OF OPHTHALMOLOGY 1996;121:19-25 19

scribed in autosomal dominant retinitis pigmentosa. Rhodopsin gene mutations have been described also in autosomal recessive retinitis pigmentosa,19,20 in a simplex case of retinitis pigmentosa,21 and in a case of congenital stationary night blindness,22 but have never previously been reported to be associated with retinitis punctata albescens. In contrast, mutations in the peripherin/RDS gene are known to be associated with a wide range of ophthalmologic phenotypes, including pattern dystrophy, fundus flavimaculatus, macular degeneration, butterfly-shaped pigment dys­trophy, and retinitis punctata albescens.23'26 Recently, Kajiwara, Berson, and Dryja27 reported three cases of digenic retinitis pigmentosa caused by mutations at the unlinked peripherin/RDS and ROM1 loci. Be­cause the mode of inheritance observed in our family was also compatible with digenism, this possibility led us to analyze the ROM1 gene. The ROM1 protein is an integral membrane protein that establishes disk shape and biogenesis on interaction with peripherin/ RDS as a heterodimer. The ROM1 gene, on chromo­some H q l 3 , is similar to the peripherin/RDS gene in its genomic structure, its photoreceptor-specific ex­pression, and in the primary structure of its protein product.28

These three genes are the only genes for which mutations have been described in association with autosomal dominant retinitis pigmentosa or digenic retinitis pigmentosa. Accordingly, we screened for mutations in the rhodopsin, peripherin/RDS, and

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iv s a Fig. 1 (Souied and associates). Pedigree of the family. Gray symbols indicate individuals affected by retinitis punctata albescens and hypercholesterolemia; solid square, an affected individual with the phenotype typical of retinitis pigmentosa; and open squares and circles, unaffected men and women, respectively. Question marks indicate individuals whose status is regarded as uncertain.

ROM1 genes in this family affected with retinitis punctata albescens.

Apolipoprotein E is a polymorphic protein that plays a central part in the metabolism of cholesterol and triglycιrides. Additionally, it plays a role in lipid transport in the nervous system29 and has a presumed relationship with autosomal dominant retinitis pig­mentosa.30

Histopathologic studies of retinitis punctata albescens are rare. We hypothesized that there is a lipidic component of these white dots deep in the retina and that this apolipoprotein could be involved in the genesis of this condition.

PATIENTS AND METHODS

WE STUDIED ONE FRENCH FAMILY AFFECTED WITH RETI-nitis punctata albescens. All affected individuals had the early-onset, severe form of retinitis pigmentosa. Early onset was defined as a decrease in visual acuity before the age of 15 years, and severe was defined as visual acuity equal to or worse than 20/200 before the age of 30 years.5 We examined the fundi of all affected members. In this family (Fig. 1), the phenotype was atypical. Patients II-3, III-2, and III-3 had retinitis punctata albescens (white dots deep in the retina and associated with findings typical of retinitis pigmento­sa31) (Fig. 2). The phenotype of patient II-5, however, was different; fundus examination disclosed classic retinitis pigmentosa but without albescent features. Optic nerve drusen were present in patient II-3 and, to a lesser degree, in patients III-2, III-3, and IV-1. Additionally, all affected members had posterior sub-capsular opacification. Individuals 1-1 and 1-2 were first cousins, and died at ages 89 and 79 years, respectively, without any symptoms of retinitis pig­mentosa (no night blindness or decreased visual acuity, according to ophthalmologic reports obtained from their physicians), and were thus considered unaffected. Fortunately, in 1990, before death, a blood sample was collected from each of them. Patient IV-1, age 16 years, had no retinal pigmenta­tion, but did have nyctalopia, decreased visual acuity, attenuated retinal vessels, waxy pallor of the optic disk, and minimal optic nerve drusen; this patient refused electroretinographic examination. Individual IV-2, age 1 year, showed no signs of retinitis pigmen-

20 AMERICAN JOURNAL OF OPHTHALMOLOGY JANUARY 1996

Fig. 2 (Souied and associates). Retinitis punctata albescens in the fundus of patient III-3.

tosa. The status of patients IV-1 and IV-2 was regarded as uncertain, and molecular analysis was not carried out on these patients for ethical reasons. Molecular analysis was performed on patients 1-1,1-2, II-3, II-5, II-6, III-2, and III-3 (Figs. 3 and 4). Individuals II-1, II-2, and II-4 did not wish to participate in our study, so no molecular analysis could be performed.

We collected 20 ml of venous blood from each affected member, using ethylenediaminetetraacetic acid (EDTA) as an anticoagulant, and we analyzed the entire coding sequences of the rhodopsin, peri-pherin/RDS, and ROM1 genes by using a combina­tion of single-strand conformation polymorphism and direct sequence analysis of each exon.32

For the rhodopsin gene, exon 1 was amplified as two partially overlapping segments so as to increase the sensitivity of single-strand conformation poly­morphism analysis.33,54 Exons 2 through 5 were each amplified as a single segment. The entire coding region, and approximately 30 base pairs of intron sequence adjacent to each of the five exons, were analyzed. Exons of the peripherin/RDS gene were analyzed in four separate reactions with four sets of primers. The first exon was amplified as two partially overlapping segments, and exons 2 and 3 were

VOL.121, No . I RETINITIS PUNCTATA ALBESCENS

amplified as a single segment. Because of the large size of the first exon with adjacent sequences (622 base pairs), we also explored it with the restriction enzyme digestion Fokl, according to the manufacturer's in­structions. The digestion products consisted of three fragments (237, 133, and 252 base pairs) that were analyzed on single-strand conformation polymor­phism gel. The entire coding region and some base pairs of the intron sequence adjacent to each of these exons were analyzed. For the ROM1 gene, exon 1 was amplified as three partially overlapping segments so as to increase the sensitivity of single-strand conforma­tion polymorphism analysis. Exons 2 and 3 were each amplified as a single segment. An intron sequence of approximately 30 base pairs adjacent to each of the three exons was analyzed.

For single single-strand conformation polymor­phism analysis, DNA (100 ng) from peripheral blood leukocytes was amplified with 0.5 μΜ of each primer and 0.1 μΐ of (alpha 33P)d-CTP (10 mCi/ml) in a 25-μ1 amplification mixture containing 10 mM Tris-HC1, pH 8.3, 50 mM KC1, 1.5 mM MgCl2, 0.2 mM dNTP, and 0.5 unit of Taq polymerase. The anneal­ing temperature was 64 C for all exons. Amplified DNA (6 μΐ) was mixed with an equal volume of formamide loading dye (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol). The samples (5 μΐ) were denatured for ten minutes at 95 C and loaded onto a polyacrylamide gel (20 X 45 X 0.04 cm Hydrolink) and electrophoresed for at least 18 hours at 4 W and at room temperature in 0.6 X Tris borate-EDTA running buffer. Gels were transferred onto Whatman paper, dried, and auto-radiographed with Kodak X-OMAT film for 16 to 48 hours. Patterns of migration on single-strand confor­mation polymorphism analysis of our patients were compared to a previous analysis of the rhodopsin, peripherin, and ROM1 genes of 70 control samples obtained from unrelated, unaffected, control sub-jects.

Exons that displayed conformational polymor­phism were sequenced on an automatic fluorometric DNA sequencer. The polymerase chain reaction fragments were excised from a low-melting-point gel (GTG NuSieve). Direct sequencing was performed on single-strand DNA, produced by asymmetric poly­merase chain reaction using the amplification primers as sequencing primers.

1 RHODOSPIN GENE MUTATION 21

Fig. 3 (Souied and associates). Single-strand conforma­tion polymorphism analysis of the rhodopsin gene (exon 2). Abnormal bands are indicated by arrows. Lane C, Control; Lanes II.3, II.5, II1.2, and III.3, Affected members of the family.

Analysis of the apolipoprotein E gene was per­formed by polymerase chain reaction and enzymatic digestion (Hhal) as described by Hixson and Verni­er.55

RESULTS

WE FOUND ONE ABNORMAL PATTERN OF MIGRATION ON

single-strand conformation polymorphism analysis in the second exon of the rhodopsin gene (Fig. 3). DNA sequence analysis showed that this abnormal band resulted from a single-base substitution: a C-to-T transition in codon 135 (Fig. 4), which resulted in a substitution of an arginine to a tryptophan amino acid in the second intracytoplasmic loop of the protein. All affected members of this family carried this mutation. The unaffected individual, 1-2, also had this mutation. The unaffected individual, II-6, did not have the mutation, but we did not analyze the other unaffected members.

22 AMERICAN JOURN/

No mutations were detected in exons 1, 3, 4, and 5 of the rhodopsin gene or in the peripherin/RDS or ROM1 genes from all individuals (data not shown).

Analysis of the apolipoprotein E gene indicated that individuals 1-1, II-3,11-4, and IH-3 had the E3/E4 haplotype, and individuals II-5 and II-6 had the E3/E3 haplotype.

DISCUSSION

WE STUDIED A FAMILY AFFECTED BY RETINITIS PUNCTATA

albescens with a mutation within the rhodopsin gene. Because the Argl35Trp mutation has been described previously to be associated with the autosomal domi­nant retinitis pigmentosa phenotype,36,57 we consid­ered this mutation to be responsible for the retinitis pigmentosa phenotype in our family. The sensitivity of single-strand conformation polymorphism for the detection of mutations in the rhodopsin, peripherin/ RDS, and ROM1 genes, with our protocol, was estimated to be between 80% and 90%. Thus, the possibility does exist that some mutations may not have been detected. Why this mutation was observed in the apparently unaffected member 1-2 is unknown. Three possible explanations are digenism, germinal mosaicism, or incomplete penetrance.

In a previous study, Souied and associates32 detect­ed the Argl35Trp mutation in the rhodopsin gene in two families in a study of 57 unrelated French families affected with autosomal dominant retinitis pigmento­sa; this mutation was not detected in 70 control samples. All affected individuals from these two families had the early-onset, severe form of autosomal dominant retinitis pigmentosa, and fundus examina­tion disclosed features typical of retinitis pigmentosa. Furthermore, this phenotype was comparable to that described previously in patients with autosomal domi­nant retinitis pigmentosa associated with an Argl35Trp mutation in the rhodopsin gene.36 This Argl35Trp rhodopsin gene mutation appears to be present in three (5%) of 58 of our subjects with autosomal dominant retinitis pigmentosa. Additional­ly, analysis of the apolipoprotein E alleles of the two affected patients in the other two families with autosomal dominant retinitis pigmentosa with the Argl35Trp mutation disclosed that they had the e3/e3 haplotype.

OF OPHTHALMOLOGY JANUARY 1996

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Fig. 4 (Souied and associates). Identification of the Argl35Trp mutation. Top, Line 1 indicates the normal amino acid sequence. Line 2 indicates the normal gene sequence of a part of exon 2 of the rhodopsin gene. An asterisk in line 3 indicates the position of the base substitution. On lines 4 and 5, abnormal gene sequences of individuals II-2 and II-5 are represented: a C-to-T substitution is observed. On line 6 is the abnormal amino-acid sequence resulting from the base substitution of arginine to tryptophan. Bottom, The automatic sequence analysis is shown for individuals II-2 and II-5. An arrow indicates the heterogeneous condition of the base substitution: the abnormal base (T, in red) was added to the normal base (C, in blue).

The mode of inheritance in the family described here was unusual. Individuals 1-1 and 1-2 were first cousins, and thus transmission of retinitis pigmentosa in generations I and II was compatible with autosomal recessive inheritance. However, analysis of the com­plete pedigree lessened the probability for such a transmission.

We have identified a rhodopsin gene mutation that is apparently associated with unusual features of retinitis pigmentosa. Hitherto, retinitis punctata albescens had been reported only in association with mutations of the peripherin/RDS gene,26 but it ap­pears that this gene is not the only one that may be implicated in retinitis punctata albescens. This par­ticular phenotype has also been described by Pearce,

Gillan, and Brosseau38 to be associated with Bardet-Biedl syndrome in an isolated northern Canadian community. The peripherin/RDS gene is not known to be involved in the Bardet-Biedl syndrome, and it seems possible that other genes could influence the albescent phenotype.

The interfamilial and intrafamilial phenotypic var­iation associated with the Argl35Trp mutation was intriguing. We hypothesized that another factor was responsible for the albescent phenotype in individuals II-3, III-2, and III-3, and that this factor could be one of the other genes implicated in autosomal dominant retinitis pigmentosa or an additional gene.

Newsome and associates39 previously described a large family with autosomal dominant retinitis pig-

VOL. 121, NO. 1 RETINITIS PUNCTATA ALBESCENS FROM RHODOSPIN GENE MUTATION 23

mentosa with abnormal clinical (optic nerve drusen) and serum lipid findings. That study led us to investigate lipid abnormalities in patients II-3, II-5, III-2, and IH-3. Hypercholesterolemia was found to be present in II-3 and III-3 but not in II-5 and III-2. Because of the absence of segregation of hypercholes­terolemia with an albescent fundus, we believe that no relationship exists between these two phenotypes.

The apolipoprotein E gene was analyzed to investi­gate the hypothesis of a genetic component for the albescent fundus in this family. The apolipoprotein E gene exists in three allelic forms, namely, E2, E3, and E4. The three isoforms (e2, e3, and e4) are present at 7%, 78%, and 14%, respectively, in the white popula­tion.40 Three reasons prompted us to analyze this gene. First, apolipoprotein E is thought to participate in the mobilization and redistribution of lipids during normal development of the nervous system and in the regeneration of peripheral nerves after injury29,41; second, Huq and associates30 reported an increased incidence of apolipoprotein E haplotypes E2/E2 and E4/E4 in retinitis pigmentosa; third, the apolipopro­tein E gene and the gene for cone-rod retinal dystro­phy have been localized to the same chromosome region.12,42,43 The genotype for patients with albescent fundus (II-3, III-2, and III-3) was E3/E4 and was E3/E3 for patient II-5. Thus, the E3/E4 haplotype did not explain the hypercholesterolemia. The presence of a high level of apoAl on lipid analysis of patients II-3 and III-3 excluded a role of apolipoprotein E in hypercholesterolemia. Because of the apparent segre­gation of allele e4 with albescent fundus, we suggest that a relationship does exist between them. However, although this observation suggests a potential role for the apolipoprotein E in retinitis punctata albescens, a significant LOD score was not possible because of the relatively small size of this family. However, it seems that, in association with the Argl35Trp mutation in the rhodopsin gene, this apolipoprotein could take part in the genesis of white dots deep in the retina.

In conclusion, the peripherin/RDS gene is not the only gene involved in the causation of retinitis punctata albescens. We found that the Argl35Trp mutation of the rhodopsin gene, known to be respon­sible for the classic phenotype of autosomal dominant retinitis pigmentosa, is associated also with retinitis punctata albescens. Two hypotheses could explain this genetic heterogeneity. First, as already described

24 AMERICAN JOURNAL OF

for the peripherin/RDS gene where mutations lead to numerous clinical phenotypes, it could be that the Argl35Trp mutation in the rhodopsin gene induces either a traditional retinitis pigmentosa or retinitis punctata albescens. Second, and because of the intrafamilial heterogeneity, it is more probable that the Argl35Trp mutation is responsible for an early-onset severe retinitis pigmentosa, and that the albes­cent phenotype is induced by a second factor, either genetic or environmental. This factor might be the e4 allele of the apolipoprotein E gene in the albescent phenotype.

REFERENCES

1. McKusick VA. Mendelian inheritance in man, 9th ed. Baltimore: Johns Hopkins University Press, 1990:974-5.

2. Bundey S, Crews SJ. A study of retinitis pigmentosa in the City of Birmingham, I: prevalence. J Med Genet 1984;21:417 -20.

3. Bunker CH, Berson EL, Bromley WC, Hayes RP, Roderick TH. Prevalence of retinitis pigmentosa in Maine. Am J Ophthalmol 1984;97:357-65.

4. Boughman JA, Conneally PM, Nance WE. Population genetic studies of retinitis pigmentosa. Am J Hum Genet 1980;32:223-35.

5. Kaplan J, Bonneau D, Frιzal J, Munnich A, Dufier JL. Clinical and genetic heterogeneity in retinitis pigmentosa. Hum Genet 1990;85:635-42.

6. Pagon KA. Retinitis pigmentosa. Surv Ophthalmol 1988;33:137-77.

7. Dryja TP, McGee TL, Hahn LB, Cowley GS, Olsson JE, Reichel E, et al. Mutations within the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa. N EnglJMed 1990;323:1302-7.

8. Farrar GJ, Kenna P, Jordan SA, Kumar-Singh R, Humphries MM, Sharp EM, et al. A three-base-pair deletion in the peripherin/RDS gene in one form of retinitis pigmentosa. Nature 1991;354:478-80.

9. Inglehearn CF, Carter SA, Keen TJ, Lindsey J, Stephenson AM, Bashir R, et al. A new locus for autosomal dominant retinitis pigmentosa on chromosome 7p. Nat Genet 1993;4:51-3.

10. Jordan SA, Farrar GJ, Kenna P, Humphries MM, Shiels DM, Kumar-Singh R, et al. Localization of an autosomal domi­nant retinitis pigmentosa gene to chromosome 7q. Nat Genet 1993;4:54-8.

11. Greenberg J, Goliath R, Beighton P, Ramesar R. A new locus for autosomal dominant retinitis pigmentosa on the short arm of chromosome 17. Hum Mol Genet 1994;3:915-8.

12. Evans K, Fryer A, Inglehearn CF, Duvall-Yong J, Whittaker JL, Gregory CY, et al. Genetic linkage of cone-rod retinal dystrophy to chromosome 19q and evidence for segregation distortion. Nat Genet 1994;6:210-3.

13. al-Maghtheh M, Inglehearn CF, Keen TJ, Evans K, Moore AT, Jay M, et al. Identification of a sixth locus for autosomal dominant retinitis pigmentosa on chromosome 19. Hum Mol Genet 1994;3:351-4.

OPHTHALMOLOGY JANUARY 1996

14· Blanton SH, Heckenlively JR, Cottingham AW, Friedman J, Sadler LA, Wagner M, et al. Linkage mapping of autosomal dominant retinitis pigmentosa (RP1) to the pericentric region of human chromosome 8. Genomics 1991;11:857-69.

15. Nathans J, Hogness DS. Isolation and nucleotide sequence of the gene encoding human rhodopsin. Proc Nati Acad Sci U S A 1984;81:4851-5.

16. Kajiwara K, Hahn LB, Mukai S, Travis GH, Berson EL, Dryja TP. Mutations in the human retinal degeneration slow gene in autosomal dominant retinitis pigmentosa. Nature 1991;354:480-3.

17. Shastry BS. Retinitis pigmentosa and related disorders: phenotypes of rhodopsin and peripherin/RDS mutations. Am J Med Genet 1994;52:467-74-

18. Yau KW. Phototransduction mechanism in retinal rods and cones: the Friedenwald Lecture. Invest Ophthalmol Vis Sci 1994;35:9-32.

19. Rosenfeld PJ, Cowley GS, McGee TI, Sandberg MA, Berson EL, Dryja TP. A null mutation in the rhodopsin gene causes rod photoreceptor dysfunction and autosomal recessive retini­tis pigmentosa. Nat Genet 1992;1:209-13.

20. Kumaramanickavel G, Maw M, Denton MJ, John S, Srikumari CR, Orth U, et al. Missense rhodopsin mutation in afamily with recessive RP [letter]. Nat Genet 1994;8:10-1.

21. Reig C, Antich J, Gean E, Garcia-Sandoval B, Ramos C, Ayuso C, et al. Identification of a novel rhodopsin mutation (Met-44-Thr) in a simplex case of retinitis pigmentosa. Hum Genet 1994;94:283-6.

22. Dryja TP, Berson EL, Rao VR, Oprian DD. Heterozygous missense mutation in the rhodopsin gene as a cause of congenital stationary night blindness. Nat Genet 1993;4: 280-3.

23. Weleber RG, Carr RE, Murphey WH, Sheffield VC, Stone EM. Phenotypic variation including retinitis pigmentosa, pattern dystrophy, and fundus flavimaculatus in a single family with a deletion of codon 153 or 154 of the peripherin/ RDS gene. Arch Ophthalmol 1993;111:1531-42.

24. Wells J, Wroblewski J, Keen J, Inglehearn C, Jubb C, Eckstein A, et al. Mutations in the human retinal degeneration slow (RDS) gene can cause either retinitis pigmentosa or macular dystrophy. Nat Genet 1993;3:213-8.

25. Nichols BE, Drack AV, Vandenburgh K, Kimura AE, Shef­field VC, Stone EM. A 2 base pair deletion in the RDS gene associated with butterfly-shaped pigment dystrophy of the fovea. Hum Mol Genet 1993;2:601-3.

26. Kajiwara K, Sandberg MA, Berson EL, Dryja TP. A null mutation in the human peripherin/RDS gene in a family with autosomal dominant retinitis punctata albescens. Nat Genet 1993;3:208-12.

27. Kajiwara K, Berson EL, Dryja TP. Digenic retinitis pigmento­sa due to mutations at the unlinked peripherin/RDS and ROM1 loci. Science 1994;264:1604-8.

28. Bascom RA, Schappert K, Mclnnes RR. Cloning of the human and murine ROM1 genes: genomic organization and sequence conservation. Hum Mol Genet 1993;2:385-91.

29. Nathan BP, Bellosta S, Sanan DA, Weisgraber KH, Mahley RW, Pitas RE. Differential effects of apolipoprotein E3 and E4 in neuronal growth in vitro. Science 1994;264:850-2.

30. Huq L, McLachlan T, Hammer HM, Bedford D, Packard CJ, Shepherd J, et al. An increased incidence of apolipoprotein E2/E2 and E4/E4 in retinitis pigmentosa. Lipids 1993;28: 995-8.

31. Heckenlively JR. Retinitis pigmentosa. Philadelphia: JB Lippincott, 1988:155-61.

32. Souied E, Gerber S, Rozet JAM, Bonneau D, Dufier JL, Ghazi I, et al. Five novel missense mutations of the rhodopsin gene in autosomal dominant retinitis pigmentosa. Hum Mol Genet 1994;3:1433-4.

33. Hash K, Andel DW. How sensitive is PCR-SSCP? Hum Mutat 1993;2:338-46.

34. Sheffield VC, Beck JS, Kwitek AE, Sandstrom DW, Stone EM. The sensitivity of single-strand-conformation polymor­phism analysis for the detection of single base substitutions. Genomics 1993;16:325-32.

35. Hixson JE, Vernier DT. Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with Hhal. J Lipid Res 1990;31:545-8.

36. Jacobson SG, Kemp CM, Sung CH, Nathans J. Retinal function and rhodopsin levels in autosomal dominant retini­tis pigmentosa with rhodopsin mutations. Am J Ophthalmol 1991;112:256-71.

37. Sung CH, Davenport CM, Hennessey JC, Maumenee IH, Jacobson SG, Heckenlively JR, et al. Rhodopsin mutations in autosomal dominant retinitis pigmentosa. Proc Nati Acad Sci U S A 1991;88:6481-5.

38. Pearce WG, Gillan JG, Brosseau L. Bardet-Biedl syndrome and retinitis punctata albescens in an isolated northern Canadian community. Can J Ophthalmol 1984;19:115-8.

39. Newsome DA, Anderson RE, May JG, McKay TA, Maude M. Clinical and serum lipid findings in a large family with autosomal dominant retinitis pigmentosa. Ophthalmology 1988;95:1691-5.

40. Davignon J, Gregg RE, Sing CF. Apolipoprotein E polymor­phism and atherosclerosis. Arteriosclerosis 1988;8:1-21.

41. Weisgraber KH, Roses AD, Strittmater WJ. The role of apolipoprotein E in the nervous system. Curr Opin Lipidol 1994;5:110-6.

42. Cladaras C, Hadzopoulou-Cladaras M, Felberg B, Pavlakis G, Zannis VI. The molecular basis of a familial apoE deficiency. J BiolChem 1987;262:2310-5.

43. Das HK, McPherson J, Bruns GA, Karathanasis SK, Breslow JL. Isolation, characterization, and mapping to chromosome 19 of the human apolipoprotein E gene. J Biol Chem 1985;260:6240-7.

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