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Genetics and Phenotypes of RPE65 Mutations in Inherited Retinal Degeneration

¹ From the Departments of Ophthalmology and Visual Sciences and ² Biological Chemistry, University of Michigan Medical School, Ann Arbor; the ³ Institute of Human Genetics, University Hospital Hamburg–Eppendorf, Germany; the ⁴ University Eye Hospital, Department II, Tübingen, Germany; the ⁵ Department of Pediatric Ophthalmology and Ophthalmogenetics, University of Regensburg, Germany; and ⁶ Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia.

	Abstract

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PURPOSE. To characterize the spectrum of RPE65 mutations present in 453 patients with retinal dystrophy with an interest in understanding the range of functional deficits attributable to sequence variants in this gene.

METHODS. The 14 exons of RPE65 were amplified by polymerase chain reaction (PCR) from patients’ DNA and analyzed for sequence changes by single-strand conformation polymorphism (SSCP) and direct sequencing. Haplotype analysis was performed using RPE65 intragenic polymorphisms. Patients were examined clinically and with visual function tests.

RESULTS. Twenty-one different disease-associated DNA sequence changes predicting missense or nonsense point mutations, insertions, deletions, and splice site defects in RPE65 were identified in 20 patients in homozygous or compound heterozygous form. In one patient, paternal uniparental isodisomy (UPD) of chromosome 1 resulted in homozygosity for a probable functional null allele. Eight of the disease-associated mutations (Y79H, E95Q, E102X, D167Y, 669delCA, IVS7+4ag, G436V, and G528V) and one mutation likely to be associated with disease (IVS6+5ga) have not been reported previously. The most commonly occurring sequence variant identified in the patients studied was the IVS1+5ga mutation, accounting for 9 of 40 (22.5%) total disease alleles. This splice site mutation, as well as R91W, the most common missense mutation, exists on at least two different genetic backgrounds. The phenotype resulting from RPE65 mutations appears to be relatively uniform and independent of mutation class, suggesting that most missense mutations (15 of 40 disease alleles [37.5%]) result in loss of function. At young ages, this group of patients has somewhat better subjective visual capacity than is typically associated with Leber congenital amaurosis (LCA) type I, with a number of patients retaining some useful visual function beyond the second decade of life.

CONCLUSIONS. RPE65 mutations account for a significant percentage (11.4%) of disease alleles in patients with early-onset retinal degeneration. The identification and characterization of patients with RPE65 mutations is likely to represent an important resource for future trials of rational therapies for retinal degeneration.

	Introduction

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The retinal pigment epithelium (RPE) performs a number of functions critical for visual processing, metabolism, and survival of the photoreceptor cells of the retina.¹ Mutations in genes expressed in the RPE have recently emerged as an important cause of inherited retinal degeneration. RPE65 was the first identified RPE-specific disease gene as a result of linkage studies that mapped the disease locus in a consanguineous family to the interval containing RPE65 on chromosome 1p31 and identified mutations responsible for childhood-onset severe retinal dystrophy.² In addition, missense mutations in RPE65 were identified in a patient with LCA type II by using the candidate gene approach.³ Since these initial reports, a wide range of disease severity has been associated with RPE65 mutations, from congenital blindness to adult-onset retinitis pigmentosa, although the most common phenotype is severe and early-onset retinal degeneration.^{4 5 6 7 8}

RPE65 encodes an abundant and evolutionarily conserved 61-kDa protein associated with the smooth endoplasmic reticulum of the RPE.^{9 10} Although the specific function of RPE65 is not yet known, a body of evidence indicates that it plays an essential role in vitamin A metabolism necessary for the synthesis of the visual pigment chromophore 11-cis retinal. Studies of Rpe65 knockout mice show that disruption of the gene results in a severely depressed electroretinogram (ERG), absence of the rhodopsin photopigment, and accumulation of all-trans retinyl esters in droplets within the RPE.¹¹ Similar ultrastructural abnormalities are also present in a strain of Swedish Briard dogs that carry a functional null allele of RPE65.^{12 13} These findings indicate that loss of RPE65 function results in a block in retinoid processing after esterification of vitamin A to membrane lipids, however, the mechanism by which RPE65 participates in retinoid isomerase activity of the RPE remains to be elucidated.

Inherited defects in vitamin A metabolism and other RPE-specific functions are likely to have a unique significance for research into the causes and treatment of retinal degeneration, since disorders affecting the RPE are, in principle, more accessible to therapeutic intervention than disorders directly affecting the proteins of the photoreceptor cells. Patients with RPE-specific defects may therefore be candidates for targeted therapies likely to become available in the near future. A necessary prerequisite for relevant clinical trials is the large-scale ascertainment and characterization of such individuals. However, few RPE65 patients have been well characterized, and the range of associated phenotypes has not been fully defined. In addition, a number of questions about the disease caused by RPE65 mutations remain unanswered, due in part to the limited amount of data on the mutation spectrum of RPE65.

We now report the results of RPE65 mutation analysis in a large collection of patients with retinal dystrophy from the United States and Europe. Our findings include the identification of a number of novel mutations. Our data are presented in the context of other known RPE65 mutations, including analysis of mutation class, prevalence, and associated phenotype.

	Methods

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Subjects
RPE65 mutation screening was performed in patients with retinal dystrophy representing diverse forms of the disease. Informed consent was obtained from the patients, according to study protocols approved by university internal review boards for human subject studies. Our research followed the tenets of the Declaration of Helsinki.

Patient Evaluation
Ophthalmic examinations of patients treated in our clinics included slit lamp biomicroscopy; assessment of visual acuity, color perception, and visual fields; electroretinograms (ERGs); and ascertainment of family history. For other patients, clinical descriptions were provided by local ophthalmologists.

Mutation Screening
DNA was prepared from whole blood using standard methods. After polymerase chain reaction (PCR) amplification of individual or groups of exons, single-strand conformation polymorphism (SSCP) analysis was used to screen for DNA sequence changes in and near gene coding regions using oligonucleotide primers and conditions published previously.^{2 14} Direct DNA sequence analysis using chain terminator cycle sequencing technology (Amersham Pharmacia Biotech, Piscataway, NJ) with the same primer pairs used for PCR amplification was used to confirm suspected DNA sequence changes, as well as for primary screening in approximately one third of cases.

Sequence Analysis
Secondary structure predictions were made using the predictive algorithms of Chou and Fasman¹⁵ and Garnier et al.^{16 17} Potential posttranslational modification sites were identified using Prosearch to scan the Prosite database.¹⁸ Coefficients of splice site efficiency were calculated according to Shapiro and Senapathy.¹⁹

Genotype Analysis
For analysis of individual mutation-associated haplotypes the microsatellite-type polymorphism located 2.8 kb upstream of the RPE65 transcription start site (locus D1S2803) was used.²⁰

	Results

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Mutation Screening
To determine the relative contribution of RPE65 mutations to the causes of inherited retinal degenerations, we screened a group of 453 unrelated patients with various forms of retinal dystrophy using a combination of SSCP and direct DNA sequencing. Our analysis resulted in the identification of 21 different disease-associated DNA sequence variants in 20 patients that predict missense or nonsense point mutations, insertion, deletion, and splice site defects in RPE65 (Table 1 , upper). These sequence variants were present in homozygous (homo) form in 7 patients and in compound (cmpd) heterozygous form in 13 patients. Disease relevance was suggested by segregation analysis (data not shown), with two notable observations. In one case (patient arRP850), two different sequence variants (1114delA and T457N) were detected on the same allele. Because 1114delA predicts a functional null mutation, the pathogenic potential of the T457N missense mutation cannot be determined. In a second case (patient 1723), segregation data were not compatible with Mendelian inheritance, in that the homozygous IVS1+5ga mutation present in the patient was found to be carried only by his father. Analysis of three DNA polymorphisms in RPE65 and 29 genetic markers spread out along both arms of chromosome 1 showed the patient to be homozygous for the paternal allele in all cases, with no inconsistencies seen for any other chromosome tested (data not shown). These findings indicate that the patient is homozygous for the IVS1+5ga mutation due to uniparental isodisomy (UPD) of chromosome 1 (Thompson D, Gal A, unpublished observations, June 2000).

Missense Mutations
The disease-associated missense mutations detected in our studies predict amino acid substitutions resulting in changes in charge (R91W, R91Q, E95Q, and D167Y), polarity (Y79H and Y368H), translation initiation (M1T), and (potentially) structure (P363T, G436V, and G528V). All missense mutations affect amino acid residues that are conserved among human, bovine, canine, rat, chicken, and salamander sequences,^{9 10 12 22 23 24} and were not present in 50 control individuals. This finding reflects the high overall conservation of the protein, ranging from 98.7% identity between humans, cattle, and dogs, to 85.7% identity between humans and salamanders. The positions of the amino acid substitutions were distributed relatively evenly throughout the length of the protein. The predicted protein structure contained a high proportion of ß-pleated sheet (in a ratio of two to one with -helical regions), with 8 of 10 missense mutations located within ß-pleated sheet domains (not shown). The amino acid substitutions did not predict major changes in overall protein folding or structure (using the Chou and Fasman or Garnier et al. algorithms^{15 16 17} ), and only substitutions at arginine-91 were predicted to perturb the local ß-pleated sheet structure (changing it to -helix). The sequence changes also did not disrupt predicted posttranslational modification sites, including consensus sequences for N-linked glycosylation, protein kinase C phosphorylation, casein kinase II phosphorylation, tyrosine kinase phosphorylation, and N-linked myristoylation. It should be noted, however, that the functional significance of these sites has not been established, and there is evidence that the mature protein contains neither O- nor N-linked glycans.²⁵ The sequence changes also did not create new donor or acceptor splice site consensus sequences with calculated splicing coefficients¹⁹ greater than or equal to the values for nearby native sites (data not shown). Thus, the effect of missense mutations is unlikely to be at the level of the transcript. Disease-associated missense mutations in this population (15 of 40 disease alleles [37.5%] occurred with slightly lower frequency than mutations predicting functional null alleles.

Null Mutations
In our studies, more than half of the disease-associated DNA sequence variants identified (11/20) predicted functional null alleles resulting from nonsense mutations (E102X, R124X, and R234X), a 1-bp insertion (144insT), small deletions (344del20, 669delCA, 831del8, and 1114 delA), and splice site mutations (IVS1+5ga, IVS7+4ag, and IVS8 + 1gt). The mutations are not clustered and are likely to result in the production of truncated protein, in some cases containing unrelated amino acid residues, or more likely, in complete absence of the protein due to greatly reduced mRNA/protein stability.

RPE65 Mutation Prevalence
In our total population of 453 patients screened, 339 were from an unselected collection of patients with retinal dystrophy, and 114 were included on the basis of a clinical diagnosis of LCA or early-onset retinal dystrophy. Of the latter 114 patients, 13 were found to have mutations in both RPE65 alleles (11.4%). Our data further show that RPE65 mutations accounted for 2.1% (7/339) of patients with autosomal recessive retinal dystrophy. The IVS1+5ga mutation was the most common of all sequence variants identified, accounting for 9 of 40 total disease alleles (22.5%). Haplotype analysis indicated that the IVS1+5ga allele occurred on at least two genetic backgrounds (Fig. 1 , lanes 1, 2, and 3). Substitutions at arginine-91 were also common, with mutations at this position occurring in four families. Analysis of these families indicated that the R91W allele also arose independently on at least two genetic backgrounds (Fig. 1 , lanes 4, 5, and 6). Among single nucleotide changes, transitions (13/23) occurred at a higher frequency than transversions (10/23). Eight of the disease-associated mutations identified in the present study (Y79H, E95Q, E102X, D167Y, 669delCA, IVS7+4ag, G436V, and G528V) and one mutation likely to be associated with disease (IVS6+5ga) have not been reported previously.

View larger version (47K): [in this window] [in a new window]   Figure 1. Genotype analysis of six patients with retinal dystrophy with RPE65 mutations IVS1+5ga or R91W using D1S2803. Alleles of the RPE65 intragenic microsatellite polymorphism D1S2803 were determined for patients with IVS1+5ga mutations: arRP713 (cmpd), arRP181 (homo), and arRP341 (cmpd) (lanes 1, 2, and 3); and for patients with R91W mutations: arRP192 (cmpd), LCA820 (homo), and arRPL (cmpd; lanes 4, 5, and 6). The data indicate that both IVS1+5ga and R91W arose independently on at least two different alleles.

	Discussion

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View larger version (16K): [in this window] [in a new window]   Figure 2. Schematic of the gene structure of RPE65 showing the positions of the 14 exons and patient mutations published to date (from References 2–8, 21, and 34), with mutations reported in the present study shown in bold.

The mechanisms by which RPE65 mutations, in general, contribute to pathogenesis are not yet known and, in part, await elucidation of the specific role of the protein in RPE physiology. Because all known missense mutations affect highly conserved residues but do not appear to disrupt protein folding or posttranslational processing and do not cluster within the linear protein sequence, it may be that these mutations inactivate a functional domain or domains present in the folded protein structure. Such mutations may be predicted to interfere with protein–protein interactions, subcellular localization, ligand binding, or intrinsic enzymatic activity necessary for the synthesis of 11-cis retinal. Each of these hypotheses will be testable when assays of the specific function(s) of the RPE65 protein become available.

Our inability to detect a second RPE65 mutant allele in three patients identified as having one probable disease-associated mutation, a general finding also reported by other groups,^{4 6 8 21} may be due to the presence of large deletions or other rearrangements undetected by current screening methods. Alternatively, mutations may occur in other regions of the RPE65 gene not analyzed in our study, including promoter and intronic regions. Mutation screening of these sequences is not practical at this time, because the critical elements that regulate RPE65 promoter activity have not yet been identified,²⁰ and no highly conserved intronic sequences beyond the splice site consensus sequences are known.²⁹ Other possibilities include dominant effect of the mutation, digenic inheritance involving the mutation of a second interacting gene,³⁰ or causal mutations in an unrelated gene. Another important issue to address in future studies is whether heterozygous individuals are at increased risk for vision loss in later life, especially in association with aging.

The severity of the disease resulting from mutations in RPE65 appears to be largely independent of the mutation types present in these patients. Previous studies have suggested that severe vision loss is the result of null mutations affecting both RPE65 alleles, whereas milder forms of disease result in cases when at least one of the two mutations is a missense allele.^{5 31} Such a correlation has also been proposed to exist for disease severity and mutations in the ATP-binding cassette transporter of rods (ABCR) gene in autosomal recessive Stargardt disease, fundus flavimaculatus, cone–rod dystrophy, and retinitis pigmentosa.³² However, our studies now show that a severe phenotype can result from a number of different combinations of RPE65 null and missense mutations. Together, these findings suggest the possibility that some missense mutations may result in true null alleles, whereas others may simply reduce the effectiveness of the protein product. Alternatively, or in addition, variability in disease severity may be determined by modifier genes that impact RPE65-related cell biology. Resolution of this issue also awaits the development of functional tests of mutant RPE65 protein.

The initial reports describing RPE65 mutations defined the associated phenotype as a childhood-onset, severe retinal dystrophy² and as LCA.³ Subsequently, it has been proposed that patients with LCA who have RPE65 mutations can be distinguished from patients who have mutations in the photoreceptor-specific guanylate cyclase gene, RetGC1, on clinical grounds.^{6 33} We find that many RPE65 patients share a common phenotype characterized by poor but useful visual function in early life (measurable cone ERGs) that declines dramatically throughout the school age years. In addition, a number of these patients retain residual islands of peripheral vision, although considerably compromised, into the third decade of life. Thus, the phenotype resulting from RPE65 mutations appears relatively uniform, possibly because each mutation exerts its effect by producing similar deficits in RPE function. It seems likely that the use of various diagnostic designations for these patients, including LCA II, early-onset severe retinal dystrophy, autosomal recessive retinal dystrophy, and early severe retinitis pigmentosa, merely reflects usage preferences by individual ophthalmologists rather than actual phenotypic differences that define patient subtypes.

The phenotype and functional defects resulting from RPE65 mutations, as well as the existence of both mouse¹¹ and canine¹² models of the disease, makes this patient population attractive candidates for future therapeutic trials focused on manipulation of the vitamin A cycle. Patients with the RPE65 mutation who have onset of disease in infancy and who retain reasonable visual function that is lost only over the course of many years would seem to be ideal subjects for therapeutic intervention. Identifying these individuals at young ages will enhance therapeutic opportunities. Research from many laboratories over the next few years will determine which of the many approaches currently under study, including gene therapy, RPE transplantation, and retinoid and survival factor therapy, may have the greatest potential for success in this group of patients. Meanwhile, in anticipation of such trials, continued characterization of this patient population, as well as the corresponding animal models, remain important goals for the immediate future.

	Acknowledgements

 
The authors thank David Hanna, Angela Cassar, and Jingtong Gao for excellent technical assistance.

	Footnotes

 
Presented in part at the annual meetings of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 1998 and 1999.

Supported by National Institutes of Health (Grants EY05627, EY06094, EY07003, EY09193), The Foundation Fighting Blindness, Research to Prevent Blindness, Daniel Matzkin Research Fund, the Deutsche Forschungsgemeinschaft Grants GA 210/12-1, SFB 430/C2, and the German and British Pro Retina Associations.

Submitted for publication April 24, 2000; revised August 2, 2000; accepted August 30, 2000.

Commercial relationships policy: N.

Corresponding author: Debra A. Thompson, University of Michigan Medical School, 533 W. K. Kellogg Eye Center, 1000 Wall Street, Ann Arbor, MI 48105-0714. dathom{at}umich.edu

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				Y. Takahashi, G. Moiseyev, Y. Chen, and J.-x. Ma The Roles of Three Palmitoylation Sites of RPE65 in Its Membrane Association and Isomerohydrolase Activity Invest. Ophthalmol. Vis. Sci., December 1, 2006; 47(12): 5191 - 5196. [Abstract] [Full Text] [PDF]

				Y. Takahashi, Y. Chen, G. Moiseyev, and J.-x. Ma Two Point Mutations of RPE65 from Patients with Retinal Dystrophies Decrease the Stability of RPE65 Protein and Abolish Its Isomerohydrolase Activity J. Biol. Chem., August 4, 2006; 281(31): 21820 - 21826. [Abstract] [Full Text] [PDF]

				J. H. Fingert, D. A. Eliason, N. C. Phillips, A. J. Lotery, V. C. Sheffield, and E. M. Stone Case of stargardt disease caused by uniparental isodisomy. Arch Ophthalmol, May 1, 2006; 124(5): 744 - 745. [Full Text] [PDF]

				Y. Chen, G. Moiseyev, Y. Takahashi, and J.-x. Ma RPE65 Gene Delivery Restores Isomerohydrolase Activity and Prevents Early Cone Loss in Rpe65-/- Mice. Invest. Ophthalmol. Vis. Sci., March 1, 2006; 47(3): 1177 - 1184. [Abstract] [Full Text] [PDF]

				G. Moiseyev, Y. Takahashi, Y. Chen, S. Gentleman, T. M. Redmond, R. K. Crouch, and J.-x. Ma RPE65 Is an Iron(II)-dependent Isomerohydrolase in the Retinoid Visual Cycle J. Biol. Chem., February 3, 2006; 281(5): 2835 - 2840. [Abstract] [Full Text] [PDF]

				J C Booij, R J Florijn, J B ten Brink, W Loves, F Meire, M J van Schooneveld, P T. de Jong, and A A B Bergen Identification of mutations in the AIPL1, CRB1, GUCY2D, RPE65, and RPGRIP1 genes in patients with juvenile retinitis pigmentosa J. Med. Genet., November 1, 2005; 42(11): e67 - e67. [Abstract] [Full Text] [PDF]

				T. M. Redmond, E. Poliakov, S. Yu, J.-Y. Tsai, Z. Lu, and S. Gentleman Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle PNAS, September 20, 2005; 102(38): 13658 - 13663. [Abstract] [Full Text] [PDF]

				J. Zernant, M. Kulm, S. Dharmaraj, A. I. den Hollander, I. Perrault, M. N. Preising, B. Lorenz, J. Kaplan, F. P. M. Cremers, I. Maumenee, et al. Genotyping Microarray (Disease Chip) for Leber Congenital Amaurosis: Detection of Modifier Alleles Invest. Ophthalmol. Vis. Sci., September 1, 2005; 46(9): 3052 - 3059. [Abstract] [Full Text] [PDF]

				M. N. A. Mandal, J. R. Heckenlively, T. Burch, L. Chen, V. Vasireddy, R. K. Koenekoop, P. A. Sieving, and R. Ayyagari Sequencing Arrays for Screening Multiple Genes Associated with Early-Onset Human Retinal Degenerations on a High-Throughput Platform Invest. Ophthalmol. Vis. Sci., September 1, 2005; 46(9): 3355 - 3362. [Abstract] [Full Text] [PDF]

				O. Strauss The Retinal Pigment Epithelium in Visual Function Physiol Rev, July 1, 2005; 85(3): 845 - 881. [Abstract] [Full Text] [PDF]

				S. G. Jacobson, T. S. Aleman, A. V. Cideciyan, A. Sumaroka, S. B. Schwartz, E. A. M. Windsor, E. I. Traboulsi, E. Heon, S. J. Pittler, A. H. Milam, et al. Identifying photoreceptors in blind eyes caused by RPE65 mutations: Prerequisite for human gene therapy success PNAS, April 26, 2005; 102(17): 6177 - 6182. [Abstract] [Full Text] [PDF]

				H. Kondo, M. Qin, A. Mizota, M. Kondo, H. Hayashi, K. Hayashi, K. Oshima, T. Tahira, and K. Hayashi A Homozygosity-Based Search for Mutations in Patients with Autosomal Recessive Retinitis Pigmentosa, Using Microsatellite Markers Invest. Ophthalmol. Vis. Sci., December 1, 2004; 45(12): 4433 - 4439. [Abstract] [Full Text] [PDF]

				H. P. N. Scholl, N. H. V. Chong, A. G. Robson, G. E. Holder, A. T. Moore, and A. C. Bird Fundus Autofluorescence in Patients with Leber Congenital Amaurosis Invest. Ophthalmol. Vis. Sci., August 1, 2004; 45(8): 2747 - 2752. [Abstract] [Full Text] [PDF]

				S. Dharmaraj, B. P. Leroy, M. M. Sohocki, R. K. Koenekoop, I. Perrault, K. Anwar, S. Khaliq, R. S. Devi, D. G. Birch, E. De Pool, et al. The Phenotype of Leber Congenital Amaurosis in Patients With AIPL1 Mutations Arch Ophthalmol, July 1, 2004; 122(7): 1029 - 1037. [Abstract] [Full Text] [PDF]

				C. L. McHenry, Y. Liu, W. Feng, A. R. Nair, K. L. Feathers, X. Ding, A. Gal, D. Vollrath, P. A. Sieving, and D. A. Thompson MERTK Arginine-844-Cysteine in a Patient with Severe Rod-Cone Dystrophy: Loss of Mutant Protein Function in Transfected Cells Invest. Ophthalmol. Vis. Sci., May 1, 2004; 45(5): 1456 - 1463. [Abstract] [Full Text] [PDF]