(RLBP1) Associated with Retinitis Punctata Albescens Evidence of Interfamilial Genetic Heterogeneity and Fundus Changes in Heterozygotes

OPHTHALMIC MOLECULAR GENETICS

SECTION EDITOR: EDWIN M. STONE, MD, PhD Novel Mutations in the Cellular Retinaldehyde–Binding Protein Gene (RLBP1) Associated With Retinitis Punctata Albescens Evidence of Interfamilial Genetic Heterogeneity and Fundus Changes in Heterozygotes

Gerald A. Fishman, MD; Mary Flynn Roberts, OD; Deborah J. Derlacki, BA; Jonna L. Grimsby, BA; Hiroyuki Yamamoto, MD, PhD; Dror Sharon, PhD; Koji M. Nishiguchi, MD; Thaddeus P. Dryja, MD

Objective: To evaluate the molecular genetic defects as- duced or absent rod responses on electroretinogram testing. sociated with retinitis punctata albescens (RPA) in 5 pa- One of the probands (patient 2:III:2) had 2 novel muta- tients from 3 families with this disease. tions in the RLBP1 gene (Arg151Trp and Gly31[2–base pair deletion], [GGA→G–]). Segregation analysis showed that Methods: We examined 3 probands and 2 clinically af- the 2 mutations were allelic and that the patient was a com- fected relatives with RPA. Clinical examinations in- pound heterozygote. Both parents of the proband mani- cluded best-corrected visual acuity, visual field testing, fested round white deposits in the retina. The other 2 pro- electroretinography, dilated fundus examination, and fun- bands had no detected pathogenic mutations in RLBP1 or dus photography. Leukocyte DNA was analyzed for mu- in the other 3 genes evaluated. tations in the exons of the genes encoding cellular retinaldehyde–binding protein 1 (RLBP1), 11-cis-retinol Conclusions: The identification of novel RLBP1 muta- dehydrogenase (RDH5), interphotoreceptor retinoid– tions in 1 of our 3 probands, all with RPA, is further evi- binding protein (RBP3), and photoreceptor all-trans- dence of genetic (nonallelic) heterogeneity in this dis- retinol dehydrogenase (RDH8). Not all patients were ease. The presence of round white deposits in the retina evaluated for mutations in each gene. The exons were may be observed in those heterozygous for RLBP1. individually amplified and screened for mutations by single-stranded conformational polymorphism analysis Clinical Relevance: Patients with a clinical presenta- or direct genomic sequencing. tion of RPA can have genetically different mutations. Dru- sen-like lesions may be observed in heterozygotes in fami- Results: The 3 probands had similar clinical findings, in- lies with this disease and a mutation in RLBP1. cluding a history of poor night vision, the presence of punctate white deposits in the retina, and substantially re- Arch Ophthalmol. 2004;122:70-75

ETINITIS PUNCTATA ALBES- pathway have been reported to cause reti- cens (RPA) is traditionally nal diseases with yellow or white retinal de- defined as an autosomal re- posits (eg, ABCA4 and RDH5),14,15 we sub- cessive disease character- sequently analyzed 3 additional genes that ized by nyctalopia, de- encode proteins in this pathway for muta- creasedR visual acuity, multiple round white tions in the 2 probands with no identified From the Department of deposits in the retina, progressive attenua- RLBP1 mutations. Ophthalmology and Visual tion of retinal arterioles, abnormal fundus Sciences, UIC Eye Center, pigmentation, progressive restriction of vi- METHODS University of Illinois at Chicago sual fields (VFs), and nondetectable or se- (Dr Fishman and Ms Derlacki), verely reduced electroretinogram (ERG) SUBJECTS and Illinois Eye Institute, amplitudes.1,2 Mutations in the gene encod- Illinois College of Optometry ing cellular retinaldehyde–binding protein This project, which involved human subjects, (Dr Roberts), Chicago; and 1(RLBP1),3-8 peripherin/RDS,9 and rhodop- conformed to the tenets of the Declaration of Massachusetts Eye and Ear sin10 have been found in patients with RPA. Helsinki and was approved by the institu- Infirmary, Harvard Medical In 3 probands with RPA, we found 1 tional review board at the University of Illi- School, Boston (Ms Grimsby nois at Chicago. We evaluated 3 probands and and Drs Yamamoto, Sharon, with 2 novel RLBP1 mutations. Because 2 clinically affected relatives with RPA. Clini- Nishiguchi, and Dryja). The RLBP1 is involved in the metabolism of vi- cal examinations included best-corrected 11-13 authors have no relevant tamin A in the retina and because de- Snellen visual acuity, dilated fundus ex- financial interest in this article. fects in genes encoding other proteins in this amina-

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©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 tion by direct and indirect ophthalmoscopy, Goldmann VFs, and ERGs. Visual field examination was performed monocu- I larly with a Goldmann perimeter using the II-2-e, II-4-e, and 12 34 V-4-e test targets. The targets were moved from a nonseeing region to a seeing region. Not all of the patients were tested × × with all of these targets. II 33 An ERG was obtained by either of 2 procedures previously 1 2 3 4 5 6 7 8-10 11-13 described.16,17 The recording techniques adhered to the international standard for clinical electroretinography established by the ××× International Society for Clinical Electrophysiology of Vision.18 III 22 All recordings were obtained with fully dilated pupils and the use 1 2 3 4 5, 6 7 8, 9 10 11 12 13 of a monopolar Burian-Allen contact lens. Scotopic responses were obtained after 40 minutes of dark adaptation. Two patients were Figure 1. Pedigree of family 1, with 3 affected individuals (proband 1:III:11 additionally tested after 17 hours of dark adaptation. [arrow] and patients 1:III:12 and 1:III:1). The affected individuals are the offspring of consanguineous unions (double horizontal lines). X indicates Three patients (1:III:11, 1:III:12, and 1:III:1) were from individuals clinically examined by us; circle, female; and square, male. one Palestinian family (family 1, Figure 1 and Table), while the remaining 2 patients (2:III:2 and 3:III:3) were from sepa- rate African American and Eastern European families (fami- tation, pigment mottling, and clumping, were also noted lies 2 and 3, respectively) (Figure 2, Figure 3, and Table). in the midperipheral retina. The retinal arterioles were not All 5 patients had night blindness in childhood, 1 (1:III:1) as attenuated. The patient’s 35-year-old mother and 36-year- early as 3 years of age and 2 (2:III:2 and 1:III:11) by 4 years of old father were each examined and found to have white age. The best-corrected visual acuities ranged from 20/20 to spots in the posterior fundus, including nasal to the optic 20/50 in all 5 patients. The spherical equivalent refractive er- disc. Both parents had vision correctable to 20/20 or bet- rors of the subjects ranged from +3.75 to −1.00 diopters. ter in each eye, and neither complained of difficulty with night vision. The number of white spots was more numer- GENETIC STUDIES ous in the mother (Figure 8), who harbored a frameshift Leukocyte DNA was analyzed for mutations in the 9 exons of mutation, than in the father, who showed more isolated RLBP1 and the 5 exons of RDH5 by single-stranded conforma- lesions and had a missense mutation. Cone and rod ERG tional polymorphism analysis and direct genomic sequencing amplitudes were normal in each parent. according to previously reported methods.7,15 We also searched The fundus examination of the proband in family 3 for mutations in the genes encoding interphotoreceptor retinoid– (3:III:3, Figure 3), age 49 years, showed a blunted foveal binding protein (RBP3) and photoreceptor all-trans-retinol de- reflex, with mild retinal arteriole attenuation and mild waxy hydrogenase (RDH8); oligonucleotide primers and polymer- pallor of the optic disc. White spots were evident pre- ase chain reaction conditions for amplifying the exons of these dominantly at and anterior to the vascular arcades 19 genes are available on the Internet. Because our 3 families did (Figure 9A). Sparse pigmentary clumping, mainly in the not show an autosomal dominant mode of transmission, we did inferior and midperipheral retina, was observed (Figure not evaluate our patients for mutations in the rhodopsin or peripherin/RDS genes. 9B). Isolated nummular lesions of chorioretinal atrophy, which were approximately three quarters of a disc diam- eter in size, were found in the inferior retina of each eye. RESULTS VISUAL FIELDS At age 21 years, the proband in family 1 (1:III:11, Figure 1) showed round punctate white deposits in the midpe- Visual field testing with a Goldmann perimeter showed ripheral retina, fewer such lesions in the posterior pole, and full VFs or only mild restriction to the II-4-e stimulus in normal optic disc, fovea, and retinal vessels. No pigmen- all 5 patients. Two of the patients (1:III:1 and 3:III:3) tary abnormalities were noted (Figure 4). Her sister showed a restriction to the II-2-e target. One patient (1:III:12, Figure 1), at age 9 years, showed multiple white (3:III:3) had central scotomas bilaterally. spots temporal to the macula in each eye, with normal optic disc and retinal vessels (Figure 5A). In addition, a pep- ELECTRORETINOGRAPHY per-like mottling was observed, most apparent in the nasal midperipheral retina (Figure 5B). A cousin (1:III:1, The dark-adapted isolated rod ERG response to a low- Figure 1), age 7 years, showed numerous white spots intensity blue stimulus was nondetectable in 4 of the 5 throughout the retina, with a normal optic disc and at- patients (1:III:1, 1:III:12, 2:III:2, and 3:III:3; Figure 10). tenuated retinal vessels. Moderate pigment granularity, par- In patient 2:III:2, this was observed even after 17 hours ticularly in the inferior retina, was also noted (Figure 6). of overnight patching. The patient’s parents each showed This patient’s 47-year-old father and 32-year-old mother normal ERG cone and rod amplitudes. One patient were each examined and found to have vision correctable (1:III:11) showed a 30% increase in the isolated rod to 20/20 in each eye. Neither showed the presence of white b-wave amplitude after 17 hours of dark adaptation com- spots in the retina of either eye. The father showed nor- pared with the amplitude after 40 minutes of dark ad- mal ERG cone and rod a- and b-wave amplitudes. aptation (Figure 11). The dark-adapted maximal ERG The fundus examination of the proband in family 2 b-wave amplitude after 40 minutes of dark adaptation was (2:III:2, Figure 2), age 7 years, showed white deposits in within 60 µV of the light-adapted high-intensity b-wave the posterior pole and midperipheral retina (Figure 7). amplitude in 4 of the 5 patients, reflecting a marked im- Retinal pigmentary abnormalities, including hypopigmen- pairment of rod function (Table).

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Family 1 Family 2 Family 3 Clinical Finding III:11* III:12 III:1 III:2* III:3* Age, y 21 9 7 7 49 Age at onset of night blindness, y 4 7 3 4 5 Best visual acuity OD 20/20+1 20/30+2 20/50+1 20/30+2 20/30 OS 20/20-1 20/30+2 20/30-2 20/25-2 20/25-1 Refractive error, spherical equivalent, diopter OD +0.12 +1.00 −0.50 +3.75 −1.00 OS Plano +1.00 +0.75 +3.67 +0.67 Electroretinogram, µV Scotopic blue 184 (40 min DA) Nondetectable Nondetectable Nondetectable Nondetectable 264 (17 h DA) (40 min DA and 17 h DA) Scotopic white 319 152 44 141 86 Photopic white 186 98 64 197 39 Photopic 32-Hz flicker 181 80 86 Not tested 38

Abbreviation: DA, dark adaptation. *Proband.

I 1 2 34 5 6 7

×× II 2 1 2, 3 4 5 6 7 8 9 ×

III 1 2 3 4 5 6 7

Figure 2. Pedigree of family 2. The arrow points to the proband (2:III:2). X indicates individuals clinically examined by us; diagonal lines, deceased; circle, female; and square, male.

Figure 4. Right fundus of proband 1:III:11, family 1. Several round punctate I white deposits are evident. The optic disc and fovea are normal, and the 1 2 3 4 retinal vessels are not attenuated.

II 33 6 3 tion analysis in the patient’s family showed that the 2 mu- 1-3 4-6 7 8 9-14 15-17 tations were allelic and that the patient was a compound ××× heterozygote (Figure 12B). The other 2 probands had no

III likely pathogenic mutations in RLBP1 detected by single- stranded conformational polymorphism analysis or direct 1 2 3 sequencing. Proband 3:III:3 was homozygous for a change in intron 7 (IVS7+20C→T) that was interpreted as likely IV 2 not to be pathogenic, because it does not appear to create 1, 2 3 45 or destroy a splice site, based on splice-site prediction soft- ware.20 The probands without identified RLBP1 muta- Figure 3. Pedigree of family 3. The arrow points to the proband. X indicates tions (1:III:11 and 3:III:3) were screened for mutations in individuals clinically examined by us; diagonal lines, deceased; circle, female; the RDH5, RDH8, and RBP3 genes. Patient 1:III:11 was ho- and square, male. mozygous for an isocoding polymorphism (Ile141Ile, ATC→ATA, 423C→A) in exon 3 of the RDH5 gene; no MOLECULAR GENETICS RDH5 sequence changes were found in patient 3:III:3. For RDH8, patient 1:III:11 was heterozygous at both of 2 bases The proband of family 2 (2:III:2) carried 2 mutations in in codon 202 (complementary DNA bases 116-117); de- RLBP1. In exon 4, there was a frameshift mutation pending on the phase of these changes, codon 202 had the (Gly31[2–base pair deletion], [GGA→G–], complemen- allelic sequences ATG (Met) and ACA (Thr), ATA (Ile), tary DNA bases 92-93delGA), and in exon 6, there was a or ACG (Thr). The alleles ATG, ACA, and ACG have been missense mutation (Arg151Trp, CGG→TGG, complemen- observed in additional patients we have evaluated in other tary DNA sequence 451C→T) (Figure 12A). Segrega- investigations of this gene, but the ATA allele has never been

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Figure 5. A, Right fundus of patient 1:III:12. There are multiple round white deposits temporal to the macula. Normal optic disc and retinal vessels are evident. B, Pigment mottling with a pepper-like appearance is most apparent in the nasal retina.

Figure 6. Right fundus of patient 1:III:1. Numerous round white deposits are Figure 7. Right fundus of proband 2:III:2, family 2. Normal retinal arterioles visible throughout the retina. Normal optic disc and retinal vessels are evident. and optic disc are evident, with porcelain white round deposits throughout the posterior pole and midperipheral retina.

encountered (data not shown). Patient 3:III:3 was heterozygous at 3 sites: Leu267Leu (CTC→CTT, 743C→T), Tyr277His (TAT→CAT, 829T→C), and Met202Thr (ATG→ACG, 116T→C). Each of these 3 variations is a non- pathogenic polymorphism in the RDH8 gene that we have found in other non-RPA individuals (data not shown). No changes in the RBP3 gene were found in patient 1:III:11 or in patient 3:III:3.

COMMENT Maw et al21 were the first to describe patients with recessive mutations in the RLBP1 gene in a retinal degenera- tion associated with diffuse “small white dots” throughout the fundus and the absence of bone spicule-like 5 Figure 8. Right fundus of proband’s mother, 2:II:5, showing moderately pigmentation. Subsequently, Burstedt et al and Morimura extensive white spots throughout the posterior fundus. et al7 described features of RPA in patients with mutations in RLBP1. Other patients with RPA from Saudi Arabia3 and Newfoundland8 have been described with mutations in atrophy in the peripheral retina, a common finding among RLBP1. In addition to the clinical findings typically found adult patients with Bothnia dystrophy,5,6 were observed in in patients with RPA, those homozygous for the RLBP1 mu- our oldest proband (3:III:3). The lesions were similar to tation Arg234Trp from northern Sweden have a predilec- those in a 52-year-old patient with RPA and an RLBP1 mu- tion to exhibit an atrophic-appearing macular lesion, par- tation described by Morimura et al.7 ticularly in those older than 30 years.5 This form of RPA Although certain phenotypic similarities exist be- has been referred to as Bothnia dystrophy.5,6 A geographic tween patients with RPA and fundus albipunctatus, as a atrophic macular lesion was not a feature observed in our group, these patients differ in certain important clinical patients with RPA; however, circular areas of geographic and genetic features. Although younger patients with

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Figure 9. Right fundus of proband 3:III:3, family 3. A, A blunted foveal reflex with mild arteriole attenuation and mild waxy pallor of the optic disc are present. Numerous round white deposits are also evident. B, In the nasal retina, pigmentary clumping and a prominent choroidal pattern inferiorly can be seen.

Normal ERG Right Eye Normal ERG Dark-Adapted Dark-Adapted Dark-Adapted 40 min 2 h 17 h

DA Blue Flash b

b a b b

V/Division b V/Division µ DA White Flash µ a a a a b b

Amplitude, 100 a Amplitude, 100 b b b

a b b LA White Flash a a a a a a 0 40 80 120 160 0 40 80 120 160 0 40 80 120 160 0 40 80 120 160 0 40 80 120 160 0 40 80 120 160 Time, ms Time, ms

Figure 10. Electroretinogram (ERG) recording from the right eye of patient Figure 11. Electroretinogram (ERG) recordings from the right eye of patient 2:III:2. There is no evidence of recordable rod function to a low-intensity 1:III:11, which show an increase in amplitude after 17 hours compared with blue-flash stimulus. DA indicates dark-adapted; LA, light-adapted; a and b, 40 minutes of dark adaptation. The letters a and b indicate a- and b-wave a- and b-wave amplitudes, respectively; and vertical bars, ranges. amplitudes, respectively; vertical bars, ranges.

either of these disorders complain of nyctalopia and typi- tion. A partial recovery in dark adaptation has also been cally show multiple round white deposits within the retina, observed in younger patients with Bothnia dystrophy. In most patients with fundus albipunctatus have a nonpro- contrast, in our patient with an RLBP1 mutation, we did gressive disease, ie, no deterioration in photoreceptor cell not observe an improvement in dark-adapted ERG re- function, no attenuation of retinal vessels, and no pig- sponses even after 17 hours of dark adaptation. ment clumping in the retina. However, other patients with Our findings suggest that the presence of a variable fundus albipunctatus and mutations in RDH5 have been number of small white spots in the fundus may be a di- described as having a cone dystrophy, leading to loss of agnostic feature observed in certain heterozygotes of this central visual acuity and reduced cone ERG amplitudes; disease. These lesions may have a phenotypic appear- these findings were most often observed in those older ance similar to drusen of the Bruch membrane. Their than 30 years.22 Particularly in younger patients with char- rather haphazard distribution, absence of a glistening (cal- acteristic features of fundus albipunctatus, cone and rod cified) appearance, and associated pigmentary changes, function can revert to normal after a prolonged period in an individual younger than 40 years, are distinguish- of dark adaptation.23 In comparison, those with RPA have ing features. Whether this finding is specific to RPA fami- a progressive loss of photoreceptor cell function and, not lies with an RLBP1 gene mutation is yet to be verified by infrequently, develop attenuated retinal vessels and pig- investigating a larger number of RPA families with or with- mentary clumping. Furthermore, mutations in different out a demonstrable RLBP1 mutation. genes have been identified in these 2 diseases. Of inter- The novel mutations in RLBP1 observed in one of est, one of our patients with RPA showed a small partial our patients, the absence of a demonstrable mutation in recovery of rod function after prolonged dark adapta- this gene in patients from 2 other families, and reports

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©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 A 1:Gly31 (2-bp del GA) 2:Arg151Trp CGG>TGG B G G A C C A T G G A C C T G T C T C T C T A G T C G G G A C A A G T C C T G T C T T T T

+ + Mutant 1/ 2/ Sequence

1/2

Asp His Gly Pro Val Ser Ser Arg Asp Lys G G A C C A T G G A C C T G T C T C T C T A G T C G G G A C A A G T

Wild-type Sequence

Figure 12. Sequences of RLBP1 mutations in proband 2:III:2. A, The top row shows the heterozygous Gly31(2–base pair [bp] deletion) and Arg151Trp mutations. Each mutant sequence is shown above the normal sequence of the same region in an unaffected individual. B, Pedigree showing that proband (arrow) is a compound heterozygote, receiving one mutant allele from each parent. Circle indicates female; square, male; and plus signs, wild-type allele.

on genetic studies in RPA patients from the existing lit- 6. Burstedt MSI, Forsman-Semb K, Golovleva I, Janunger T, Wachtmeister L, Sandgren O. Ocular phenotype of Bothnia dystrophy, an autosomal recessive reti- erature further underpin the genetic heterogeneity of RPA. nitis pigmentosa associated with an R234W mutation in the RLBP1 gene. Arch Genetic screening may be necessary to discriminate be- Ophthalmol. 2001;119:260-267. tween some patients with RPA and those with fundus al- 7. Morimura H, Berson EL, Dryja TP. Recessive mutations in the RLBP1 gene encoding cellular retinaldehyde–binding protein in a form of retinitis punctata albipunctatus, especially in those with early stages of RPA. bescens. Invest Ophthalmol Vis Sci. 1999;40:1000-1004. Genetic heterogeneity exists in both of these disorders. 8. Eichers ER, Green JS, Stockton DW, et al. Newfoundland rod-cone dystrophy, an early-onset dystrophy, is caused by splice-junction mutations in RLBP1. Am J Hum Genet. 2002;70:955-964. Submitted for publication April 1, 2003; final revision re- 9. Kajiwara K, Sandberg MA, Berson EL, Dryja TP. A null mutation in the human ceived August 15, 2003; accepted August 25, 2003. peripherin/RDS gene in a family with autosomal dominant retinitis punctata albescens. Nat Genet. 1993;3:208-212. This study was presented in part at the annual meet- 10. Souied E, Soubrane G, Benlian P, et al. Retinitis punctata albescens associated ing of the Association for Research in Vision and Ophthal- with the Arg135Trp mutation in the rhodopsin gene. Am J Ophthalmol. 1996; mology; May 6, 2002; Ft Lauderdale, Fla. 121:19-25. 11. Saari JC, Bredberg DL, Noy N. Control of substrate flow at a branch in the visual This study was supported by funds from The Founda- cycle. Biochemistry. 1994;33:3106-3112. tion Fighting Blindness, Owings Mills, Md; Grant Health- 12. Crabb JW, Carlson A, Chen S, et al. Structural and functional characterization of care Foundation, Chicago, Ill; grants EY01792 and EY08683 recombinant human cellular retinaldehyde–binding protein. Protein Sci. 1998; 7:746-757. from the National Institutes of Health, Bethesda, Md; and 13. Stecher H, Gelb MH, Saari JC, Palczewski K. Preferential release of 11-cis- an unrestricted departmental grant from Research to Pre- retinol from retinal pigment epithelial cells in the presence of cellular retinaldehyde– vent Blindness, New York, NY. binding protein. J Biol Chem. 1999;274:8577-8585. 14. Allikmets R, Singh N, Sun H, et al. A photoreceptor cell–specific ATP-binding Corresponding author: Gerald A. Fishman, MD, De- transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy partment of Ophthalmology and Visual Sciences, UIC Eye [published correction appears in Nat Genet. 1997;17:122]. Nat Genet. 1997;15: Center, University of Illinois at Chicago, Mail Code 648, 236-246. 15. 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