Four Novel Mutations in the RPE65 Gene in Patients With Leber Congenital Amaurosis

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1 HUMAN MUTATION Mutation in Brief #432 (2001) Online MUTATION IN BRIEF Four Novel Mutations in the RPE65 Gene in Patients With Leber Congenital Amaurosis Marcia J. Simovich 1, Beverly Miller 1, Hany Ezzeldin 1#, Bryan T. Kirkland 1, Genevieve McLeod 1, Chere Fulmer 1, Jeremy Nathans 2, Samuel G. Jacobson 3, and Steven J. Pittler 1*# 1 Department of Biochemistry & Molecular Biology, University of South Alabama College of Medicine, Mobile, AL, USA; 2 Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 3 Department of Ophthalmology, Scheie Eye Institute, Philadelphia, PA, USA. *Correspondence to: Dr. Steven J. Pittler, University of Alabama at Birmingham, Vision Science Research Center, Birmingham, AL , USA; Tel: ; Fax: ; pittler@uab.edu #Present address: University of Alabama at Birmingham, Vision Science Research Center, Birmingham, AL Contract grant sponsor: The Foundation Fighting Blindness, Baltimore, MD, USA Communicated by Mark H. Paalman Leber congenital amaurosis (LCA) is a heterogeneous disorder representing the congenital forms of retinitis pigmentosa accounting for about 5% of all retinal dystrophies. The RPE65 gene product is required for regeneration of the visual pigment for phototransduction. Defects in the RPE65 gene have so far been shown to account for ~10 % of known cases of LCA. Here we describe four additional novel mutations in the RPE65 gene (c.889dela, c.131g>a, c.1249g>c, c.430t>g) and several novel polymorphisms in a large series of LCA patients Wiley-Liss, Inc. KEY WORDS: Leber congenital amaurosis; LCA2; mutation screening; RPE65; retina pigment epithelium; photoreceptor. INTRODUCTION Leber congenital amaurosis (LCA) is the name given to a group of heterogeneous autosomal recessive disorders that severely affect vision in infants. Cardinal features of the disease are markedly reduced visual performance at earliest testing, nystagmus, and absent or greatly reduced electroretinogram at birth. Limited, but significant association with neurologic disturbance or keratoconus has been observed. The relationship between LCA and early onset retinitis pigmentosa, which manifests a few years after birth, remains unclear. The first gene identified in which defects were shown to be causally related to LCA was guanylate cyclase type I (GUCY2D, MIM# ; (Perrault et al., 1996). To date 6 additional loci have been reported and four of the genes have been identified (LCA3, MIM# ; LCA5, MIM# ; Crx, MIM# ; RPE65, MIM# ; AIPL1, MIM# ; and TULP1, MIM# ). The subject of this study, the RPE65 gene consists of 14 exons encompassing an open reading frame of 533 codons (Hamel et al., 1993; Nicoletti et al., 1995; Morimura et al., 1998). While the function of the retina pigment epithelium specific RPE65 protein has not been unequivocally established it clearly is required for the regeneration of 11-cis retinal in the visual cycle (Redmond & Hamel, 2000). Absence of the protein product in mice leads to a Received 14 March 2001; revised manuscript accepted 10 May WILEY-LISS, INC.

2 2 Simovich et al. buildup of all-trans retinyl esters and a progressive retinal degeneration (Redmond et al., 1998). Marlhens et al., 1997 first reported a defect in the RPE65 gene in a patient with LCA; defects were also reported in patients with early onset retinitis pigmentosa (Gu, et al., 1997). MATERIALS AND METHODS Patient samples Ninety-eight unrelated patients diagnosed with LCA were included in the study. Informed consent was obtained from all patients analyzed. Peripheral blood was collected in an EDTA tube and used for isolation of genomic DNA with a commercially available kit (Qiagen, Valencia, CA or Quantum Biotechnologies, Carlsbad, CA), or by equilibrium centrifugation in cesium chloride. Each of the 14 exons of the RPE65 gene was amplified by PCR with Taq DNA polymerase and 1.5 mm MgCl 2 under standard PCR conditions using the following primer sets. Primer pairs for exons 1-3, 9, 10, 14 as well as sense primers for exons 4, 7, and 11, and antisense primers for exon 5 were made and used at temperatures described (Gu et al., 1997). The exon 12 primer pair, exon 5 sense, and exons 4 and 11 antisense primers were synthesized and used as described (Marlhens et al., 1997). Primers 6F 5 -CTCTCAACTGGAGGACATTC; 6R 5 -TCAGAAGAGGACAGATTGGT; 7R 5 -AAAGGTAGGCAAAGC- AAATC; 8F 5 -CAGCCCTTTCATTCACAAGC; 8R 5 -TCTTCAGAATCACAAACTTG; and 13F 5 -GCATAT- TGACTGATTGCTTG, 13R 5 -GCAGTAAGAAGAGTATTCAG were synthesized and used at 60, 55, 60, and 55 degrees annealing temp, respectively. Mutation analysis by track DNA sequencing Each PCR amplification product was assessed for purity on an agarose gel and then purified using a Qiagen Qiaquick PCR purification kit. Purified DNA was cycle sequenced in batches with only one dideoxynucleotide and electrophoresed through 6% acrylamide gels. Autoradiographs were examined for band alterations, which were confirmed by standard DNA cycle sequencing. All potential DNA sequence changes were verified by DNA sequence analysis on both strands from at least two independent PCR products. Once confirmed, sequence changes were further characterized wherever possible, by segregation analysis in family members of the affected patients. Additionally, none of the likely pathogenic changes were found in more than 200 control samples analyzed. RESULTS Ninety-eight patients were examined for mutations in each of the 14 exons of the RPE65 gene resulting in the identification of seven different likely polymorphisms including silent codon changes and intron changes outside of donor and acceptor splice sites (Table 1). One polymorphism (c.1056g>a) has been frequently observed in several diverse populations (Redmond & Hamel, 2000). Table 1: Novel polymorphic non-pathogenic changes. No. of patients Exon/Intron DNA change 1 Amino acid 1 3 c.231c>a V c.1155g>a T c.1056g>a E c.609c>t A c.570c>t Y190 1 I13 c c>t -- 1 I1 c.11+34t>a -- 1 numbering based on nomenclature system of Antonarakis et al., 1998 Eleven likely pathogenic changes in 8 different patients were identified (Table 2). None of the likely pathogenic changes were found in more than 200 control samples analyzed.

3 Mutations in the RPE65 Gene 3 Patient 33 was heterozygous for a single nucleotide deletion that causes a frameshift that would truncate at K297 and end with a C-terminal extension of 26 amino acids. No second site mutation was found and segregation could not be followed because the patient was adopted and biological parents could not be traced. Patient 35 was heterozygous for two missense mutations leading to a change of arginine to glutamine at pos. 44 and asparagine to lysine at pos The change at pos. 321 was also found in an unrelated patient previously reported (Lotery et al., 2000). Co-segregation of the mutations was found in the patients mother, father and two siblings. Table 2: Likely pathogenic changes identified in RPE65 in LCA patient samples. Patient Exon/intron DNA change + Consequence Allele status Co-Segregation c.889dela frameshift heterozygous CND c.131g>a R44Q heterozygous Yes c.963t>g N321K heterozygous Yes c.1249g>c E417Q homozygous Yes 57 2 I1 c.11+5g>a Splicing? homozygous Yes c del frameshift homozygous Yes c.118g>a G40S heterozygous Yes c.544c>t H182Y heterozygous Yes 84 5 c.430t>g Y144D homozygous Yes c.272g>a R91Q heterozygous Yes 91 4 I7 c.725+4a>g Splicing? heterozygous Yes +numbering based on nomenclature system of Antonarakis et al., novel mutations found in this study are in boldface 2 previously reported by Lotery et al., previously reported by Morimura et al., previously reported by Thompson et al., could not be determined A homozygous missense mutation was found in patient 38 resulting in a glutamate to glutamine change at pos Both parents were heterozygous for the observed change. Patient 57 was homozygous for an intron change that has been previously reported (Lotery et al., 2000) and has been found in an unrelated patient (Gu et al., 1997). Patient 60 was homozygous for a 20 bp deletion in exon 4, which results in a frameshift and a truncated protein at E104 that is extended 25 amino acids. This change has been previously reported (Van Hooser et al., 2000; Lotery et al., 2000). Both the mother and father and two siblings show consistent co-segregation. Patient 76 was a compound heterozygote for missense mutations causing a glycine to serine change at pos. 40 and a histidine to tyrosine change at pos This change and segregation analysis was previously reported (Morimura et al., 1998). Patient 84 was homozygous for a missense mutation leading to a histidine to aspartate change at pos Cosegregation of the mutation was observed in the patient s mother and sister. Patient 91 is a compound heterozygote for missense and intron changes that cause an arginine to glutamine change at pos. 91 and a nucleotide change in the consensus donor splice site. The adenosine residue at position +4 in the donor site is conserved in 68 % of genes that have been examined and is present in 11 of 13 of the RPE65 gene donor sites. Co-segregation was verified in both parents and one other affected sibling. These mutations were previously reported (Thompson et al., 2000). DISCUSSION The prevalence of RPE65 mutations causative for LCA has been estimated from 6.8 to 16 % (Gu et al., 1997; Marlhens et al., 1997; Morimura et al., 1998; Lotery et al., 2000; Thompson et al., 2000). Our study identified 8 probands with RPE65 pathogenic changes of 98 examined (8.2 %). Combining the data from all of these studies suggests that RPE65 defects account for approximately 10 % of LCA. All of the affected individuals identified in this study were either homozygous or compound heterozygous for disease causing mutations except patient 33

4 4 Simovich et al. where a second site mutation could not be found. It is possible that in addition to the 1 bp deletion found in this patient, that a promoter or unidentified intron mutation exists. Another possibility is that the truncated protein that could be produced acts in a dominant fashion to inhibit function of the normal protein. The precise role of RPE65 has not been determined, however, it is clear that the protein is required for production of 11-cis retinal and visual pigment regeneration. One current hypothesis is that RPE65 acts as a scaffolding protein that binds retinoid and presents it to the retinal isomerase for conversion from the all-trans form back to the 11-cis form. The distribution of mutations that cause disease may be useful in identifying important functional domains. As shown in Figure 1, 56 % of the reported mutations in the protein coding region are clustered within 4 exons (3, 4, 9, 10), each containing 6 or more mutations. X Missense Nonsense Insertion Deletion Splice donor X X X X X This report Novel Reported elsewhere Figure 1. Spectrum of RPE65 mutations reported to date ACKNOWLEDGMENTS This work was supported by a grant from the Foundation Fighting Blindness to SJP and SGJ, Lions/USA Eye Research Institute to SJP, NIH EY and the Daniel Matzkin Research Fund to SGJ. We thank Ms. E. DeCastro, Mr. David Hanna, and Ms. J. Emmons for clinical coordination. ELECTRONIC DATABASE INFORMATION The RPE65 cdna reference sequence can be obtained under accession number NM_ RPE65 genomic sequences can be found in the GenBank database (U20476-U20488 and U20510; AF AF039868). REFERENCES Antonarakis SE, Nomenclature Working Group (1998) Recommendations for a nomenclature system for human gene mutations. Human Mutation 11:1-3 Gu SM, Thompson DA, Srikumari CR, Lorenz B, Finckh U, Nicoletti A, Murthy KR, Rathmann M, Kumaramanickavel G, Denton MJ, Gal A (1997) Mutations in RPE65 cause autosomal recessive childhood-onset severe retinal dystrophy. Nat. Genet. 17: Hamel CP, Tsilou E, Pfeffer BA, Hooks JJ, Detrick B, Redmond TM (1993) Molecular cloning and expression of RPE65, a novel retinal pigment epithelium-specific microsomal protein that is post- transcriptionally regulated in vitro. J. Biol. Chem. 268: Lotery AJ, Namperumalsamy P, Jacobson SG, Weleber RG, Fishman GA, Musarella MA, Hoyt CS, Heon E, Levin A, Jan J, Lam B, Carr RE, Franklin A, Radha S, Andorf JL, Sheffield VC, Stone EM (2000) Mutation analysis of 3 genes in patients with Leber congenital amaurosis. Arch. Ophthalmol. 118: Marlhens F, Bareil C, Griffoin JM, Zrenner E, Amalric P, Eliaou C, Liu SY, Harris E, Redmond TM, Arnaud B, Claustres M, Hamel CP (1997) Mutations in RPE65 cause Leber's congenital amaurosis [letter]. Nat. Genet. 17:

5 Mutations in the RPE65 Gene 5 Morimura H, Fishman GA, Grover SA, Fulton AB, Berson EL, Dryja TP (1998) Mutations in the RPE65 gene in patients with autosomal recessive retinitis pigmentosa or leber congenital amaurosis. Proc. Natl. Acad. Sci. USA 95: Nicoletti A, Wong DJ, Kawase K, Gibson LH, Yang-Feng TL, Richards JE, Thompson DA (1995) Molecular characterization of the human gene encoding an abundant 61 kda protein specific to the retinal pigment epithelium. Human Molec. Genet. 4: Perrault I, Rozet JM, Calvas P, Gerber S, Camuzat A, Dollfus H, Chatelin S, Souied E, Ghazi I, Leowski C, Bonnemaison M, Le Paslier D, Frezal J, Dufier JL, Pittler S, Munnich A, Kaplan J (1996) Retinal-specific guanylate cyclase gene mutations in Leber's congenital amaurosis. Nat. Genet. 14: Redmond TM, Hamel CP (2000) Genetic analysis of RPE65: from human disease to mouse model. Meth. Enzymol. 316: Redmond TM, Yu S, Lee E, Bok D, Hamasaki D, Chen N, Goletz P, Ma JX, Crouch RK, Pfeifer K (1998) Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle. Nat. Genet. 20: Thompson DA, Gyurus P, Fleischer LL, Bingham EL, McHenry CL, Apfelstedt-Sylla E, Zrenner E, Lorenz B, Richards JE, Jacobson SG, Sieving PA, Gal A (2000) Genetics and phenotypes of RPE65 mutations in inherited retinal degeneration. Invest. Ophthalmol.Vis. Sci. 41: Van Hooser JP, Aleman TS, He YG, Cideciyan AV, Kuksa V, Pittler SJ, Stone EM, Jacobson SG, Palczewski K (2000) Rapid restoration of visual pigment and function with oral retinoid in a mouse model of childhood blindness. Proc. Natl. Acad. Sci. USA 97: