Analysis of Three Genes in Leber Congenital Amaurosis in Indonesian Patients

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Vision Research 43 (2003) 3087–3093 www.elsevier.com/locate/visres

Analysis of three genes in Leber congenital amaurosis in Indonesian patients

Rita S. Sitorus a,b,*, Birgit Lorenz a, Markus N. Preising a a Department of Paediatric Ophthalmology, Strabismology and Ophthalmogenetics, Klinikum, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany b Department of Ophthalmology, School of Medicine, University of Indonesia, Salemba 6, Jakarta 10430, Indonesia Received 23 June 2003

Abstract Purpose. To assess the frequency, the pattern of disease causing mutations, and phenotypic variations in patients with Leber congenital amaurosis (LCA) from Indonesia. Patients and methods. Twenty-one unrelated index cases with a clinical diagnosis of LCA were screened for mutations in the coding sequence of RetGC1, RPE65 and AIPL1 gene with single strand conformation polymorphism analysis followed by direct sequencing and restriction enzyme digestion. Results. Four novel disease causing mutations were identified: Three in the RPE65 gene (106del9bp, G32V and Y435C) in two of 21 index cases and one in the AIPL1 (K14E). Two of them were homozygous and one was compound-heterozygous. No disease causing mutation was identified in RetGC1. Conclusions. The four novel disease causing mutations identified in this study confirmed the diagnosis of LCA which has not been recognized before in Indonesia. The frequency of RPE65 mutations was 9.5%; and of AIPL1 mutations 4.8%. This was in general accordance with previous studies reported from other countries. Unlike in those studies, no disease causing RetGC1 mutations could be identified in our patients. Phenotypically, the RPE65 and AIPL1 mutations identified in this study caused nearly total blindness by the second decade of life, but had a different onset of symptoms. The patients with the RPE65 mutations retained some useful visual function until the end of the first decade, which progressed to total blindness during the second decade of life, whereas the (homozygous) AIPL1 mutation was associated with nearly total blindness from infancy on. Therefore, RPE65 mutations have to be considered to cause early onset severe retinal degeneration (EOSRD), and AIPL1 mutations a form of LCA. 2003 Elsevier Ltd. All rights reserved.

Keywords: Leber congenital amaurosis LCA; RPE65; RetGC1; AIPL1; Mutation; Genotype–phenotype

1. Introduction LCA is generally inherited in an autosomal recessive manner although some autosomal dominant families Leber congenital amaurosis (LCA, MIM 204000, have been described. To date, mutations in six genes 204100) is a term used to refer to a group of inherited have been reported to cause a subset of LCA. These retinal dystrophies characterized by severe, bilateral include GUCY2D or RetGC1, encoding retinal guany- visual impairment from birth (Leber, 1869). LCA ac- late cyclase (Perrault et al., 1996); RPE65, encoding a counts for at least 5% of all retinal dystrophies and is retinal pigment epithelium-speciﬁc 65 KD protein one of the main causes of blindness in children (Hec- (Dharmaraj et al., 2000; Felius et al., 2002; Hamel, kenlively, 1988). Griﬀoin, Lasquellec, Bazalgette, & Arnaud, 2001; Lorenz et al., 2000; Lotery et al., 2000; Marcos, Ruiz, Borrego, & Antinolo, 2001; Marlhens et al., 1998; * Corresponding author. Permanent address: Department of Oph- Morimura et al., 1998; Poehner et al., 2000; Simovich thalmology, School of Medicine, University of Indonesia, Salemba 6, et al., 2001; Thompson et al., 2000); CRX, encoding the Jakarta - 10430, Indonesia. Fax: +62-21-3927516. E-mail addresses: [email protected], [email protected] cone-rod homeobox-containing gene (Freund et al., gensburg.de (R.S. Sitorus), [email protected] 1997); AIPL1 (Sohocki et al., 2000), CRB1 (Lotery (M.N. Preising). et al., 2001) and RPGRIP (Dryja et al., 2001). A severe

0042-6989/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.visres.2003.08.008 3088 R.S. Sitorus et al. / Vision Research 43 (2003) 3087–3093 early-onset retinal dystrophy with features suggesting exons of RPE65, RetGC1 and AIPL1 genes using oli- LCA has been described with a mutation in the TULP1 gonucleotide primers previously reported (RetGC1, gene, encoding the Tubby-like protein 1 (Hagstrom, Perrault et al., 1996 and RPE65, Marlhens et al., 1997). North, Nishina, Berson, & Dryja, 1998). The identifi- AIPL1 primers have been designed by the authors using cation of these disease causing mutations raised the the VECTOR NTI 7.0 Suite (VECTOR NTI Suite 7.0 possibility of performing specific genotype–phenotype (2001)). Subsequently, PCR products were analysed correlations that could be used to provide more accurate using single strand conformation polymorphism (SSCP) diagnostic and prognostic information to the patients. analysis. The PCR products presenting with aberrant Ethnic differences and geographic variations may electrophoretic patterns were subjected to direct se- affect the frequencies and pattern of mutations. So far quencing using the fluorescent chain termination tech- molecular genetic studies of LCA are generally based on nique. To confirm the sequencing result, the PCR Caucasian populations. As Indonesian ophthalmolo- products of the patients, their relatives and controls gists do not satisfactorily perform the diagnosis of LCA, were analyzed using restriction endonuclease assay when no data concerning LCA in Indonesian patients have possible. been reported before. On the other hand recent data show that LCA patients carrying defects in the RPE65 gene are expected to be candidates for future therapeutic trials. Consequently for that reason, characterization of 3. Results patients is an important issue to be explored. There is a need to study this disease in Indonesian 3.1. Molecular results patients by identifying the kind and frequency of sequence variations present in the Indonesian population, We identified two disease-causing mutations in performing genotype–phenotype correlations and to RPE65 and one in AIPL1 mutation of the 21 index cases discover new sequence changes with clinical impact. studied. These mutations are summarized in Table 1. This study was undertaken to identify and describe Mutations in RetGC1 were identified but could not be mutations in three genes (RetGC1, RPE65 and AIPL1) proven with any degree of certainty to be disease-caus- in Indonesian patients with LCA. ing. In addition, a number of benign sequence variants within the RPE65, RetGC1 and AIPL1 genes were identified (Table 2). Most of these variants identified were intronic. 2. Patients and methods

Twenty-one unrelated individuals from The School 3.2. RPE65 gene mutation of Blind and Paediatric Ophthalmology Clinic, Cipto Mangunkusumo Hospital University of Indonesia, We identified a homozygous 9 bp in-frame deletion at clinically diagnosed by two of the authors (RS and BL) nucleotide 106 (106del9bp) predicted to abolish three with LCA were screened for coding sequence mutations amino acids (LWL36del) in case 1 (II:2 in Fig. 1A). in RetGC1, RPE65 and AIPL1. Electroretinograms A missense mutation, G32V was identified in exon 3 (ERGs) and visual field tests could not be performed of RPE65 in case 2 (II:4 in Fig. 1B) at nucleotide 95. due to the limited facilities available. This mutation affects the first base pair of exon 3 and is Blood samples were taken after informed consent considered to have an impact on splicing. The G32V according to The Declaration of Helsinki from affected mutation was confirmed using the TaaI-restriction en- individuals and, if available, from their relatives as well. zyme. In the compound allele, an A fi G transition at DNA was extracted from peripheral blood lymphocytes nucleotide position 1304 in exon 12 was identified according to (Miller, Dykes, & Polesky, 1988). DNA leading to the substitution of tyrosine by cysteine at samples were subjected to PCR to amplify the coding amino acid position 435. The Y435C mutation creates a

Table 1 Probable disease-causing mutations identified in patients with Leber congenital amaurosis Case no. Gene Nucleotide Mutation Exon Restriction digest Base change 1 RPE65 106 106del9bpa 3 – CCCCTCTGGCTCACC fi CCCACC homozygous 2 RPE65 95 G32Va 3 TaaI gGC fi gTCb 1304 Y435Ca 12 Hpy8I TAT fi TGT 3 AIPL1 42 K14Ea 1 XmnIAAA fi AGA homozygous a Novel. b Lower case letters indicate intronic sequences. R.S. Sitorus et al. / Vision Research 43 (2003) 3087–3093 3089

Table 2 Benign sequence variants in Indonesian patients Variant Base change Exon Allele frequency Previously reported RPE65 Glu352Glu 1056G fi A 10 13/44 Morimura et al. (1998) IVS3-46g/a 246-46G fi A 4 4/16 IVS6 + 72a/g 643 + 72A fi G 6 2/8 IVS6-33g/c 645–33G fi C 7 12/68 IVS12-33a/c 1341–33A fi C 13 1/8 Marcos et al. (2001) IVS13 + 45t/c 1450 + 45T fi C 13 2/8 RetGC1 P701S 2174C fi T 10 19/50 Dharmaraj et al. (2000) C457C 1373C fi T 4 6/6 IVS13 + 74c/t 2576 + 74C fi T 13 2/2 30UTR 3528T fi C 20 55/56 Dharmaraj et al. (2000) AIPL1 L100L 300A fi G 3 13/22 Sohocki et al. (2000) P217P 651G fi A 5 40/66 Sohocki et al. (2000) IVS2-10a/c 277–10A fi C 3 29/32 Sohocki et al. (2000) IVS2-14g/a 277–14G fi A 3 1/21 Sohocki et al. (2000) IVS3 + 16g/c 465 + 16GC 3 1/21 IVS3 + 28g/a 465 + 28G fi A 3 2/21

No other family members were available for genetic testing in both families. So, segregation analysis was not possible.

3.3. AIPL1 gene mutation

The K14E mutation in AIPL1 is a novel mutation. The A fi G substitution at the first nucleotide of codon 14 in exon 1 leads to the substitution of a glutamine residue for a lysine residue. The K14E mutation coseg- regated in the family in an autosomal recessive pattern. The K14E mutation creates a new XmnI restriction site (Fig. 2B). It was not present in any of 60 unaffected individuals or in other probands tested.

3.4. Clinical results

Fig. 1. (A) Pedigree of Family 1 with case 1 (II:2) shown to carry the Fig. 1 shows the pedigrees, phenotypes and genotypes 106del9bp mutation in RPE65; (B) pedigree of Family 2 with case 2 of the families carrying RPE65 mutations. In Fig. 2 the (II:4) carrying two mutations in RPE65. Patients available for genetic same data are summarized for the patients carrying the testing and clinical examination are indicated by an arrow. (C) TaaI AIPL1 mutation. Table 3 summarizes the clinical find- restriction digest for the heterozygous G32V mutation. The mutation ings. Family members available for clinical examination creates a novel restriction site as shown by the amplimer of II.2 which shows fragments at 249 and 202 bp; (D) Hpy8I restriction digest for the will be described in the following. heterozygous Y435L mutation. The mutation creates a novel restric- Case 1. The 106del9bp mutation in RPE65 was tion site as shown by the 232 bp restriction fragment of the amplimer identified in case 1 (II:2 in Fig. 1A), a 21 year-old pa- of II.4 which is cut into two fragments of 122 and 101 bp and (E) tient. She was a child of a consanguineous marriage of fundus photograph of case 2 (II:4) at age 23 years; C––control and unaffected parents, and had two affected brothers. She M––marker. had a history of life long severe vision impairment. When examined at age 21, her visual acuity was restricted to the detection of hand movements in both new Hpy8I restriction site. The sequencing results were eyes. Fine nystagmus was noted and the anterior seg- confirmed by restriction digest. ments were normal. Funduscopy showed attenuated All three mutations were not present in any of 50 vessels and pigment clumps in the peripheral retina. control individuals or in other probands tested. Fundus photographs of her were not available. 3090 R.S. Sitorus et al. / Vision Research 43 (2003) 3087–3093

Family 3. The homozygous K14E mutation in the AIPL1 gene was found in a female, 22 years of age (II:3 in Fig. 2A). From birth, the parents of II:3 had noted an inability to move alone. However, systemic diseases were absent. Her visual acuity was insufficient to attend normal school since childhood and at the time of examination at the age of 22 years her it was restricted to hand movement and light perception. While the anterior segments were normal there were attenuated retinal vessel, hypopigmentation and bone spicule-like pigment deposits in the midperipheral retina (Fig. 2C/D). The unrelated parents (I:1 and I:2) were both unaffected with normal anterior segments. While the father presented with a small macular drusen, the optic nerve head, fovea and retinal vessels were normal in both eyes. Her sister aged 6 years (II:6 in Fig. 2A) was affected. The parents noted that this young girl had a marked visual impairment since birth. She could not follow objects and was unable to attend the school as a normal child. The digitoocular sign of Franceschetti (eye poking) was ob- Fig. 2. (A) Pedigree of Family 3 with AIPL1 mutation K14E. The served. Visual acuity was limited to hand movements in index case is indicated by an arrow; (B) XmnI restriction digest for the both eyes, and the anterior segments were normal. Due homozygous K14E mutation. The mutation creates a novel restriction to the presence of marked pendular-jerk nystagmus site as shown by the restriction fragments of 152 and 103 bp from the fundus photography was not possible. 255 bp amplimer; C––control and M––marker. Fundus photograph of case 3 (II:3) at age 22 years; (C) right eye and (D) left eye. 4. Discussion Case 2. The compound heterozygous mutation G32V/Y435C in RPE65 was found in case 2 (II:4 in Fig. This study is the first to be performed in patients from 1B), a 23 year-old male. He completed normal school Indonesia diagnosed with LCA, which has previously until age 9, by which time visual impairment had sig- been an under diagnosed, if not disregarded entity in nificantly progressed. At the age of 18 severe visual Indonesia. impairment was diagnosed. At the time of the examin- The prevalence of causative mutations of RPE65 for ation the visual acuity was limited to light perception in LCA has been estimated from 3.0% to 15.6% (Dhar- both eyes. Fine nystagmus was noted, and the anterior maraj et al., 2000; Lotery et al., 2000; Morimura et al., segments were within the normal limit. Funduscopy 1998; Simovich et al., 2001) although early and recurring showed attenuated retinal vessels, moderate pallor of the reports have already put forward that RPE65 mutations optic disc, areas of pigment epithelium atrophy and cause an early onset severe retinal degeneration pigment clumps in the mid peripheral retina. In the (EOSRD) which is phenotypically different from LCA macula and the surrounding superotemporal retina is- (Gu et al., 1997; Lorenz et al., 2000; Thompson et al., lands of marked hyperpigmentations were observed 2000). The important difference of EOSRD and LCA is a (Fig. 1E). significantly better initial visual performance in EOSRD

Table 3 Clinical findings Case Age [y] Age at onset [y] Fundus Nystagmus Visual acuity Consanguinity 1 21 >3 Attenuated vessels, +, mild-moderate HM, OU Yes pigment clumps 2 23 9 Fig. 1E +, mild-moderate LP, OU No Case 3, Family 3, 22 Birth Fig. 2C and D +, severe HM, LP No (II:3) Case 3, Family 3, 9 Birth Not possible +, pendular-jerk HM, OU No (II:6) HM ¼ Hand movements; LP ¼ Light perception; OU ¼ both eyes. R.S. Sitorus et al. / Vision Research 43 (2003) 3087–3093 3091 promising sufficient residual vision to attend regular cases of EOSRD (Felius et al., 2002; Gu et al., 1997; schools until the early teen years. AIPL1 mutations were Lorenz et al., 2000). In this present study a preserved identified in 7–9% of LCA cases worldwide. visual function up to the age of about 9 years was noted Our study was performed on a small set of index in cases carrying RPE65 mutations, which afterwards cases. The frequencies of mutations identified in RPE65 progressed to almost total blindness during the second (9.5%) and in AIPL1 (4.8%) are in general accordance decade of life. Unfortunately we could not get any de- with previous studies (Dharmaraj et al., 2000; Lotery tailed information concerning the visual performance of et al., 2000; Morimura et al., 1998; Simovich et al., 2001; these patients in early childhood as described by another Sohocki et al., 2000). Our findings, however, were in studies (Lorenz et al., 2000). It is suggested that visual contrast with a study in the Indian population, which performance might be better during the first year of life, reported that RPE65 seems to be less involved in the which may reflect postnatal physiological cone matura- causation of LCA (Joseph et al., 2002). All index cases tion (Lorenz et al., 2000). carrying the mutations were students at a School for Clinical characterisation of mutations in RPE65 and Blind in Bandung with a total number of about 200 other RPE expressed genes involved in retinol metabo- blind persons caused by different etiologies. Most of lism suggest that impairment of the ocular metabolism them come from West Java region with a total popula- of retinol predominantly affects rods (Hamel et al., tion of 35,000,611 in an area of 44,355 km2. 2001). Our findings are in agreement with a mutation of RPE65 resulting in degeneration of the macular as 4.1. RPE65 mutations previously suggested, as changes in the macular may reflect the greater density of rods over cones at the pe- The precise role of RPE65 has not been determined, riphery of the macula. however, it is clear that the protein has a crucial role in the metabolism of vitamin A in the visual cycle (Red- 4.2. AIPL1 mutations mond et al., 1998). Regeneration of the visual pigment is performed in the RPE, and requires the isomerisation of AIPL1 has been reported to cause LCA4 (Sohocki all trans-retinol to 11-cis-retinol, followed by the oxi- et al., 2000). It is a cytoplasmic protein with similarities dation to 11-cis-retinal. In the absence of RPE65, the to aryl hydrocarbon receptor-interacting protein (AIP), isomerisation process is blocked, leading to an accu- a member of the FK-506-binding protein family. It mulation of all-trans-retinyl esters (Redmond et al., contains three tetratricopeptide repeats (TPR), a motif 1998). The distribution of disease causing mutations that is found in proteins with nuclear transport or pro- may be useful in identifying important functional do- tein chaperone activity, suggesting that AIPL1 may be mains. A most complete collection of RPE65 mutations involved in retinal protein folding or trafficking (So- can be obtained from the World Wide Web (http:// hocki et al., 2000). However, the exact function of www.retina-international.org/sci-news/rpe65mut.htm). AIPL1 remains unknown. Only a few data on AIPL1 The 106del9bp mutation in index case 1 has not been mutations providing detailed clinical information were reported previously. It predicts an in-frame mutation reported so far. In the present study cases II:3 and II:6 and synthesis of a polypeptide shortened by three amino (Family 3) are homozygous for a K14E mutation. Ly- acids L36, W37 and L38. The amino acids W37 and L38 sine at this position is highly conserved in human, rat are conserved throughout the vertebrates from Homo- and mouse. It is likely, therefore, that this substitution sapiens down to the Japanese firebelly newt Cynops represents a disease causing-mutation in this family. pyrrogaster, whereas L36 is not. It is very likely that Our data suggest that AIPL1 mutations cause a se- 106del9bp is predicting a functional null allele. vere, early-onset blindness, a condition that can be dis- The compound heterozygous mutation G32V/Y435C tinguished from blindness in patients with the RPE65 identified in case 2 cannot be evaluated according to the mutation (LCA2). In both affected index cases (II:3 and affected protein domains since no domains have been II:6 in Family 3) total blindness was noted from infancy described in RPE65. However, Gly 32 and Tyr 435 are onwards. Pendular-jerk nystagmus and eye poking conserved residues throughout the vertebrates from confirmed a severe perturbation of the development of Homosapiens down to the Japanese firebelly newt visual function during the early stages of life. The severe, Cynops pyrrogaster. It is presumed therefore, that these early-onset of blindness was also observed in our sepa- substitutions will lead to functional null alleles. rated study in Caucasian patients harboring AIPL1 Correlations between LCA genotypes and the disease mutations (manuscript in preparation). This severe early severity have been established for patients with RetGC1 onset of blindness may be due to a defect in the regu- (LCA1) and RPE65 mutations (LCA2) (Dharmaraj lation of cell cycle progression during photoreceptor et al., 2000; Dharmaraj et al., 2000; Perrault et al., 1999). maturation. Thus, our data support that AIPL1 plays a Several publications reported homozygous or com- role during early retinal development. It has been hy- pound heterozygous RPE65 mutations associated with pothesized that this gene is important in cytosolic 3092 R.S. Sitorus et al. / Vision Research 43 (2003) 3087–3093 stability and/or nuclear transport of NUB1 during reg- tients with RPE65 mutation retain useful visual func- ulation of cell cycle progression during photoreceptor tions until the first decade, which is progressing to total maturation (Akey et al., 2002). blindness in the second to third decade of life. The pa- The phenotype of AIPL1 mutations might be vari- tient with the AIPL1 mutation suffered from almost able. Recently, it was reported that mutations in AIPL1 complete impairment of vision since birth. It is neces- cause autosomal dominant cone-rod dystrophy (So- sary, however, to conduct molecular screening studies in hocki et al., 2000). AIPL1 mutations in our case segre- a larger scale on extended Indonesian families, to allow gated in an autosomal recessive pattern. Concerning the better recognition of pathogenicity of certain alleles and progression we were not able to distinguish between genotype–phenotype correlations. cone-rod and rod-cone progression. Indeed recent results by van der van der Spuy et al. (2002) indicate a rod- specific expression of AIPL1 which argues against a Acknowledgements cone-rod progression. As AIPL1 and RetGC1 were assigned closely on The authors thank all the patients and families for 17p13, both AIPL1 and RetGC1 should be screened for their participation in this study; to Eijkman Molecular mutations in families with affected individuals whose Biology Institute Jakarta for the possibility to isolate the LCA locus maps to 17p13, or in families with affected DNAs; L. Simangunsong MD, S. Suhardjono, MD, The individuals who are homozygous for mutations in either School of Blindness, Bandung, Indonesia for providing gene, unless linkage excludes one of the genes by intra- contact to the patients. D. Purba MD and Andhi- genic markers (Sohocki et al., 2000). In our study the Jakarta Eye Center and G. Schuch for the excellent presence of RetGC1 mutation could be excluded by the fundus photographs; to Dr John Wellard for the valu- mutational screening. able comments to the manuscript. RS spent periods of research in Germany, supported by a Grant of The 4.3. RetGC1 mutations Georg Forster Research Fellowship Program (The Al- exander von Humboldt Stiftung Special Program). Mutations in RetGC1 gene have been identified in a large number of LCA families with the mutation frequency of 6.0–20.3% (Lotery et al., 2000). (Dharmaraj References et al., 2000; Perrault et al., 2000) Our study could not identify any disease causing mutations in RetGC1, de- Akey, D. T., Zhu, X., Dyer, M., Li, A., Sorensen, A., Blackshaw, S., spite having screened the entire open reading frame of Fukuda-Kamitani, T., Daiger, S. P., Craft, C. M., Kamitani, T., & the gene. This may be due to the difference in the ethnic Sohocki, M. M. (2002). The inherited blindness associated protein background between Indonesian patients and previously AIPL1 interacts with the cell cycle regulator protein NUB1. Human Molecular Genetics, 11(22), 2723–2733. studied populations that had been recruited from Dharmaraj, S., Silva, E., Li, Y. Y., Loyer, M., Pina, A. L., Sunness, J., countries surrounding the Mediterranean Sea (Hanein Koenekoop, R. K., & Maumenee, I. H. (2000). Modifying factors et al., 2002). This introduces a bias in studies on patients in Leber congenital amaurosis. Investigative Ophthalmology and from elsewhere. Visual Science, 4, S196. In the present study we identified a frequent mutation Dharmaraj, S. R., Silva, E. R., Pina, A. L., Li, Y. Y., Yang, J. M., Carter, C. R., Loyer, M. K., El Hilali, H. K., Traboulsi, E. K., (P701S) in 19 of 50 alleles in the heterozygote state in- Sundin, O. K., Zhu, D. K., Koenekoop, R. K., & Maumenee, I. H. cluding case 2, in whom a compound heterozygote (2000). Mutational analysis and clinical correlation in Leber RPE65 mutation was identified as disease-causing mu- congenital amaurosis. Ophthalmic Genetics, 21(3), 135–150. tation. The P701S was reported previously as sequence Dryja, T. P., Adams, S. M., Grimsby, J. L., McGee, T. L., Hong, D. variant which did not co-segregate with both of the af- H., Li, T., Andreasson, S., & Berson, E. L. (2001). Null rpgrip1 alleles in patients with leber congenital amaurosis. Amercian fected probands in the family, but was detected only in Journal Human Genetics, 68(5), 1295–1298. the worse affected sibling in the pedigree (Dharmaraj Felius, J., Thompson, D. A., Khan, N. W., Bingham, E. L., Jamison, J. et al., 2000). Further studies will be necessary to confirm A., Kemp, J. A., & Sieving, P. A. (2002). Clinical course and visual whether this variation significantly alters the protein function in a family with mutations in the RPE65 gene. 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