![]() Received 26 August 2004 | Accepted 7 January 2005 | Published 15 April 2005 |
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Characterization of the Crumbs homolog 2 (CRB2) gene and analysis of its role in retinitis pigmentosa and Leber congenital amaurosis
José A. J. M. van den Hurk,1 Penny Rashbass,2
Ronald Roepman,1 Jason Davis,3 Krysta E. J. Voesenek,1
Maarten L. Arends,1 Marijke N. Zonneveld,1 Marga H. G. van
Roekel,1 Karen Cameron,2 Klaus Rohrschneider,4 John R.
Heckenlively,5 Robert K. Koenekoop,6 Carel B. Hoyng,7
Frans P. M. Cremers,1
Anneke I. den Hollander1
Departments of 1Human Genetics and 7Ophthalmology, Radboud Univerisity Nijmegen Medical Center, Nijmegen, the Netherlands; 2Center for Developmental Genetics, Department of Biomedical Science, University of Sheffield, Sheffield, UK; 3Molecular and Cellular Biochemistry, Department of Biochemistry, University of Oxford, Oxford, UK; 4Universität-Augenklinik, Ruprecht-Karls-Universität, Heidelberg, Germany; 5Department of Ophthalmology, University of Michigan, Ann Arbor, MI; 6Children's Vision Center, McGill University, Montreal, Canada
Correspondence to: Anneke I. den Hollander, Department of Human Genetics, 417 Radboud Univerisity Nijmegen Medical Center, P. O. Box 9101 6500 HB, Nijmegen, the Netherlands; Phone: (+31) 24.3614017 FAX: (+31) 24.3540488; email: a.denhollander@antrg.umcn.nl
Abstract
Purpose: Mutations in the Crumbs homolog 1 (CRB1) gene cause autosomal recessive retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). Database searches reveal two other Crumbs homologs on chromosomes 9q33.3 and 19p13.3. The purpose of this study was to characterize the Crumbs homolog 2 (CRB2) gene on 9q33.3, to analyze its expression pattern, and to determine whether mutations in CRB2 are associated with RP and LCA.
Methods: The CRB2 mRNA and its expression pattern in human tissues were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The cellular expression of Crb2 in the mouse eye was determined by mRNA in situ hybridizations. The open reading frame and splice junctions of CRB2 were analyzed for mutations by single-strand conformation analysis and direct nucleotide sequencing in 85 RP patients and 79 LCA patients.
Results: The CRB2 gene consists of 13 exons and encodes a 1285 amino acid transmembrane protein. CRB2 is mainly expressed in retina, brain, and kidney. In mouse retina Crb2 expression was detected in all cell layers. Mutation analysis of the CRB2 gene revealed 11 sequence variants leading to an amino acid substitution. Three of them were not identified in control individuals and affect conserved amino acid residues. However, the patients that carry these sequence variants do not have a second sequence variant on the other allele, excluding autosomal recessive inheritance of CRB2 sequence variants as a cause of their disease.
Conclusions: This study shows that CRB2 sequence variants are not a common cause of autosomal recessive RP and LCA. It is possible that a more complex clinical phenotype is associated with the loss or altered function of CRB2 in humans due to its expression in tissues other than the retina.
Introduction
Mutations in the Crumbs homolog 1 (CRB1) gene cause severe retinal dystrophies. CRB1 mutations have been found in patients with retinitis pigmentosa (RP) type 12 [1-3], an early onset form of autosomal recessive RP characterized by a preserved para-arteriolar retinal pigment epithelium (PPRPE). CRB1 mutations have also been detected in patients with early onset RP without PPRPE but with other RP12 characteristics [2,4], and in RP patients who had developed Coats-like exudative vasculopathy, a relatively rare complication of RP characterized by vascular abnormalities, yellow extravascular lipid depositions, and in severe cases retinal detachment [5]. In addition, mutations in the CRB1 gene have been detected in 10-13% of patients with Leber congenital amaurosis (LCA), the most severe retinal dystrophy leading to blindness or severe visual impairment in the first year of life [5-7].
CRB1 is homologous to the Drosophila transmembrane protein Crumbs [1]. Crumbs is a key regulator of epithelial apical-basal polarity in Drosophila [8,9], and is essential for the correct formation of the photoreceptors [10,11]. Crumbs is a central component of a molecular scaffold that controls assembly of the zonula adherens. Crumbs and CRB1 localize to corresponding subdomains of the photoreceptor apical plasma membrane: the stalk of Drosophila photoreceptors and the inner segment of mammalian photoreceptors, respectively [10].
Database searches with the 37 amino acid cytoplasmic domain of Drosophila Crumbs revealed two other human homologs [12]. The characterization of the Crumbs homolog 3 (CRB3) gene on 19p13.3 was recently described [13]. Here, we report the characterization of the Crumbs homolog 2 (CRB2) gene, located on chromosome 9q33.3. We demonstrate that CRB2 is predominantly expressed in retina, brain, and kidney. Several examples of homologous genes that are implicated in similar diseases have been described in the literature, such as fibrillin-1 and -2 in microfibrillopathies [14], and fibulin-5 and -6 in age related macular degeneration [15,16]. In view of its homology to CRB1 and its expression in the retina, we considered CRB2 to be a candidate gene for retinal dystrophies and searched for CRB2 gene mutations in 85 RP patients and 79 LCA patients.
Methods
Bioinformatics
Protein and translated nucleic acid sequence databases were searched for Crumbs homologs with the C-terminal 37 amino acids of Drosophila Crumbs and human CRB1, using the BLAST programs [17]. The hits that were generated were used in a BLAT search of the UCSC Human Genome Working Draft [18] to obtain corresponding genomic fragments and annotated gene predictions (FGenesH++, Genscan, Twinscan, and GeneID). Deduced protein sequences were searched for functional patterns using SMART [19] and RPSBLAST [17], and by comparison to the CRB1 and Crumbs domain structures. Multiple sequence alignments were created using ClustalW [20] and boxed with BoxShade 3.21.
Reverse transcription (RT)-PCR analysis
To verify the CRB2 gene prediction, four overlapping nested primer sets were designed from the predicted sequence and used for RT-PCR on human retina cDNA (Table 1). The primary PCR reactions were performed on 2.5 μl human retina Marathon-Ready cDNA (Clontech, Palo Alto, CA) with the GC-rich PCR System (Roche, Almere, Netherlands) according to the manufacturer's recommendations. Primary PCR products were purified (Qiaquick Gel Extraction Kit, Qiagen, Venlo, Netherlands), diluted 100x, and used as a template for the nested PCR reactions, using the GC-rich PCR System.
For expression profile analysis of the CRB2 gene, total RNA samples from seven human tissues (brain, liver, lung, skeletal muscle, placenta, heart, and kidney) were purchased from Clontech. Total RNA from retina, fetal eye, RPE/choroid, and RPE cell line ARPE-19 [21] was isolated using RNAzolB (Campro Scientific, Veenendaal, Netherlands). Total fetal cochlea RNA was isolated by CsCl purification [22]. Total RNA samples were treated with DNase I (Invitrogen, Breda, Netherlands). Randomly primed cDNA synthesis and RT-PCR were performed as described previously [23]. Table 2 shows primers used to determine the expression profile for CRB1, CRB2, and GAPDH. Amplification was performed with a touchdown PCR [24] at 94 °C for 1 min, Tann for 1 min, 72 °C for 1 min using annealing temperatures (Tann) of 59 °C for 2 cycles, 58 °C for 2 cycles, 57 °C for 2 cycles, 56 °C for 2 cycles, and 55 °C for 24 cycles.
In situ hybridizations
A digoxigenin labeled antisense riboprobe was transcribed from a mouse Crb2 cDNA clone containing the last 1 kb of coding sequence and 2.5 kb of 3' untranslated region. This clone was identified by sequencing random clones from a custom made cDNA library of dissected E9.5 mouse optic primorida (P. Rashbass, unpublished data).
Retinas from adult CBA mice were fixed overnight in 4% paraformaldehyde, transferred to 30% sucrose in phosphate buffered saline (PBS) prior to embedding in OCT, where they were rapidly frozen on dry ice. 15 μm cryostat sections were cut and air dried. Slides were hybridized overnight at 65 °C with the diluted riboprobe in hybridization buffer (50% formamide, 5% SSC pH 7.0, 0.5% CHAPS, 1 mg/ml yeast RNA, 5 mM EGTA, 50 μg/ml heparin, 2% Roche blocking reagent). They were then washed at 65 °C in 50% formamide, 5x SSC pH 4.5, 0.1% SDS followed by 2 washes of 50% formamide, 2x SSC pH 4.5, 0.1% SDS. Following 3 washes in Tris buffered saline Tween (TBST) they were blocked in 10% sheep serum in TBST and incubated in anti-digoxigenin-AP conjugated Fab fragment (1:2000, Roche) overnight. Crb2 RNA localization was determined using NBT/BCIP detection (Roche) of alkaline phosphatase. Once color had developed sufficiently, the sections were refixed in 4% paraformaldehyde prior to imaging using DIC optics (Olympus BX60 microscope with Spot-TR color camera [Diagnostic Instruments. Inc., Sterling Heights, MI] attached). Images were prepared for publication using Adobe Photoshop.
Mutation analysis
This study involved human subjects and was approved by the institutional review board of the Radboud Univerisity Nijmegen Medical Center. Mutation analysis of the CRB2 gene was performed in 79 unrelated patients with autosomal recessive or isolated LCA, 81 unrelated patients with autosomal recessive or isolated RP and 4 unrelated patients with autosomal dominant RP. Of the patients with autosomal recessive or isolated RP, four presented with PPRPE, six had developed Coats-like exudative vasculopathy, and two were diagnosed with kidney disease (possible Senior-Löken syndrome).
For mutation analysis of the open reading frame and splice junctions of CRB2, 27 primer pairs were designed (Table 3). Exons 2, 3, and 8.1-8.2 were screened for alterations by sequence analysis and the remaining amplicons by single-strand conformation analysis (SSCA). In 10 patients, all amplicons of the CRB2 gene were analyzed by sequencing. PCR reactions for SSCA were performed in a volume of 10 μl using 25 ng of genomic DNA and 2 pmol of each primer labeled with 0.2 pmol [γ-32P]ATP. PCR reactions for sequencing purposes were performed in a volume of 25 μl using 100 ng of genomic DNA and 10 pmol of each primer. PCR reactions contained 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.5 U Taq DNA polymerase and PCR buffer provided by the manufacturer (Invitrogen). For amplification of exons 3, 5, and 10, 1 M DMSO was added to the reaction mixture. Amplification was performed with a touchdown PCR at 94 °C for 20 s, Tann for 20 s, 72 °C for 30 s using Tann of 66 °C for 3 cycles, 64 °C for 3 cycles, 62 °C for 3 cycles, and 60 °C for 24 cycles (for SSCA) or 30 cycles (for sequencing). PCR amplification was preceded by 3 min denaturing at 94 °C, and concluded with 3 min elongation at 72 °C. For SSCA, PCR products were denatured and electrophoresed on 8% polyacrylamide gels at 4 °C (10-12 W for 16 h). PCR products with altered electrophoretic mobility were analyzed by sequencing. For DNA sequencing, PCR products were purified with the StrataPrep 96 PCR Purification Kit (Stratagene, La Jolla, CA). Sequencing was performed with BigDye Terminator chemistry (version 3) on a 3730 or 3100 DNA Analyzer (Applied Biosystems, Foster City, CA).
Amino acid substitutions p.V97L and p.E187D were analyzed in control individuals with the amplification-refractory mutation system (ARMS) [25,26]. For p.P46L and p.A351T ARMS was unsuccessful, while diagnostic restriction sites were not available. The remaining amino acid substitutions were tested in control individuals by PCR amplification and restriction enzyme digestion. Sequence variant p.P116L was analyzed with EagI; p.M145T with BsmAI; p.G159A with HaeIII; p.R534Q with SfcI, p.R610W with HpaII; p.H746Q with BsgI, and p.T1110M with NspI. Restriction enzymes were purchased from Invitrogen or New England Biolabs (Beverly, MA), and were used according to the recommendations of the manufacturers.
Results
Characterization of the CRB2 gene and analysis of its expression pattern
Web based database searches with the 37 amino acid C-terminal cytoplasmic domains of Drosophila Crumbs and human CRB1 identified two possible homologs on chromosomes 9q33.3 and 19p13.3. The identification of the Crumbs homolog 3 (CRB3) gene on 19p13.3 was recently described [13]. Gene predictions of the Crumbs homolog 2 (CRB2) gene on 9q33.3 were verified by RT-PCR on human retina cDNA. The CRB2 gene consists of 13 exons, encoding a 1285 amino acid transmembrane protein. CRB2 has a similar protein structure as CRB1, characterized by a signal peptide, three laminin A globular (G)-like domains flanked by epidermal growth factor (EGF)-like domains, a transmembrane domain, and a 37 amino acid cytoplasmic domain (Figure 1A).
Laminin A G-like domains and EGF-like domains are extracellular protein modules that can interact with extracellular proteins and components of the extracellular matrix [27-30]. Laminin A G-like domains consist of 158-180 amino acid residues, exhibit low overall homology, but have some residues that are highly conserved [31]. EGF-like domains typically consist of six cysteine residues that interact with each other by the formation of disulfide bridges. These stabilize the native fold, which comprises a major and minor β-sheet. Disulfide bridges are formed between the first and third cysteine residues, the second, and fourth residues, and the fifth and sixth residues [32]. The CRB2 protein contains 17 EGF-like domains, however the first and thirteenth EGF-like domains are truncated, since they contain only 4 cysteine residues (Figure 1A). A distinct subgroup of EGF-like domains has been identified that contains a consensus sequence associated with calcium binding (cb) [33]. In other proteins that contain tandemly repeated cbEGF domains, such as fibrillin-1, Ca2+ is predicted to rigidify the interdomain region resulting in a rod-like structure [34]. In CRB2, EGF-like domains 3-9 contain a calcium binding sequence (Figure 1A).
The C-terminal cytoplasmic domains of Crumbs, CRB1, CRB2, and CRB3 are highly conserved (Figure 1B). The cytoplasmic domain of CRB2 is 68% identical to that of Drosophila Crumbs, 49% identical to CRB1, and 59% identical to CRB3. This domain is of crucial importance, since it has been shown to link Crumbs homologs to several cytoplasmic proteins [12,35-42].
The expression pattern of the CRB2 gene was determined by RT-PCR on RNA samples from 11 human tissues and RPE cell line ARPE-19. CRB1 and CRB2 are both expressed in retina, fetal eye and brain. However, CRB2 is also expressed in kidney, ARPE-19, RPE/choroid, and at low levels in lung, placenta, and heart (Figure 2). The cellular expression of the Crb2 gene in the adult mouse eye was analyzed by mRNA in situ hybridizations. Crb2 is strongly expressed in the outer nuclear layer, containing the cell bodies of the photoreceptor cells, and in the inner nuclear layer, containing the cell bodies of the horizontal, bipolar, amacrine, and Müller glial cells (Figure 3A). Furthermore, some cells in the ganglion cell layer also express Crb2 (Figure 3B). The number of cells stained in the ganglion cell layer varies throughout the retina, suggesting that this particular subset of cells may be displaced amacrine cells rather than ganglion cells.
Mutation analysis of the CRB2 gene in patients with retinitis pigmentosa and Leber congenital amaurosis
Mutation analysis of the CRB2 ORF and splice junctions in 85 RP patients and 79 LCA patients revealed 14 sequence variants in the untranslated regions and introns, 14 synonymous codon changes, and 11 amino acid substitutions (Table 4). To determine whether the amino acid substitutions are pathogenic, their frequency was analyzed in control individuals (Table 5). In addition, we determined whether these amino acid changes are conservative or nonconservative with respect to charge, size, and polarity [43], the likelihood that the amino acids involved are replaced during evolution (Blosum62 score) [44], and whether they are conserved in homologous domains of Crumbs, CRB1, and CRB2 (Table 5). The amino acid substitutions p.M145T, p.G159A, and p.T1110M are common polymorphisms, since they were found frequently on alleles of RP and LCA patients and control individuals (Table 4 and Table 5). Substitutions p.R610W and p.H746Q do not affect conserved residues in the CRB2 protein (Table 5), and both amino acid substitutions were found on 0.5-1% of control alleles (Table 5). Therefore, it is likely that they represent rare, nonpathogenic sequence variants.
Four amino acid substitutions, p.V97L, p.P116L, p.E187D, and p.R534Q were not found on more than 340 control alleles, and the presence of two amino acid changes, p.P46L and p.A351T, could not be determined by ARMS (Table 5). These six sequence variants were all found in different patient samples; p.P46L, p.V97L, p.P116L, and p.E187D were found in LCA patients, p.A351T was identified in an isolated RP patient, and p.R534Q was detected in a patient with autosomal dominant RP. The latter patient, however, appeared to have a causative mutation in one of the known autosomal dominant RP genes (A. Bergen, personal communication). To determine whether the four LCA patients and the isolated RP patient carried a second CRB2 sequence variant on the other allele, all CRB2 exons and their splice junctions were sequenced in these patients. No additional sequence changes were identified, strongly suggesting that CRB2 sequence variants are not associated with RP or LCA through an autosomal recessive inheritance pattern.
The amino acid substitutions p.V97L and p.A351T affect nonconserved residues in the EGF-like domains (Figure 4 and Figure 5). Substitution p.V97L is a conservative (aliphatic) amino acid change, while p.A351T is a nonconservative change that is likely to occur during evolution (neutral negative Blosum62 score; Table 5). However, this does not necessarily rule out pathogenicity, since not all CRB1 mutations affect conserved residues and not all missense mutations are nonconservative changes [45].
The amino acid substitutions p.P116L and p.E187D affect conserved residues located in the cbEGF-like domains (Figure 4). Sequence variant p.P116L is a nonconservative amino acid substitution changing a proline to an aliphatic amino acid, and has a Blosum score of -3, which indicates that proline is not likely to be replaced by leucine during evolution [44]. However, in one of 30 cbEGF-like domains of Crumbs, CRB1 and CRB2, this residue is also aliphatic (isoleucine, Figure 4). The substitution p.E187D is a conservative amino acid change, since both glutamic acid and aspartic acid are negatively charged polar residues. In one of 30 cbEGF-like domains of Crumbs, CRB1 and CRB2, this residue is an aspartic acid (Figure 4). Substitution p.P46L changes an amino acid that is conserved in the non-cbEGF-like domains, and is located at a similar position as p.P116L. In one of 36 non-cbEGF-like domains of Crumbs, CRB1 and CRB2, this residue is a leucine (Figure 5). Based on these findings pathogenicity of the amino acid changes p.E187D, p.P116L, and p.P46L is not very likely, but cannot completely be ruled out.
Discussion
Humans have three homologs of Drosophila Crumbs, CRB1 on 1q31.3, CRB2 on 9q33.3, and CRB3 on 19p13.3. The protein structures of Crumbs, CRB1, and CRB2 are highly similar, characterized by a large extracellular domain consisting of laminin A G-like domains flanked by EGF-like domains, and a 37 amino acid cytoplasmic domain. CRB3 encodes a small protein of 120 amino acids lacking the laminin A G-like domains and EGF-like domains, but it has a cytoplasmic domain that is highly similar to that of Crumbs, CRB1, and CRB2 [13]. The CRB1 gene encodes a transmembrane and an extracellular protein isoform by alternative splicing [1,46]. Gene predictions suggest that the CRB2 gene may also be alternatively spliced, encoding a transmembrane and an extracellular protein isoform, but this was not analyzed by RT-PCR [47].
All three Crumbs homologs are expressed in the retina [1,13,48]. CRB1 expression was found to be restricted to retina and brain [1,49], although in mouse Crb1 expression was also detected in kidney, stomach, lung, and testis [50]. However, CRB2 and CRB3 are expressed in a wider range of tissues. In this report we showed that CRB2 is expressed in brain, kidney, ARPE-19, RPE/choroid, and at low levels in lung, placenta, and heart. CRB3 is expressed in lung, kidney, colon, and mammary gland [13]. In mouse retina Crb1 expression is restricted to the photoreceptors and some cells of the inner nuclear layer [49], while Crb2 is expressed in all layers of the retina. In the photoreceptors, the Crb1 protein is confined to the inner segment, where it concentrates just apical to the outer limiting membrane (OLM), a region of cell-cell adhesion between photoreceptors and Müller cells [10]. Recently, it was shown that Crb2 and Crb3 colocalize with Crb1 at the OLM [48]. Redundant functions of CRB1, CRB2, and CRB3 are not known, but their co-localization at the OLM, and the high similarity of their cytoplasmic domains, suggest a possible overlap or competition in function [48].
Mutations in the CRB1 gene cause autosomal recessive RP and LCA [1,5,6]. We screened the CRB2 gene for mutations in patients with RP and LCA and detected 11 amino acid substitutions. Five of them (p.M145T, p.G159A, p.R610W, p.H746Q, p.T1110M) were detected in control individuals, and are therefore likely to be nonpathogenic sequence variants. One sequence variant, p.R534Q, was detected in a patient with autosomal dominant RP, but this patient had a causative mutation in a known autosomal dominant RP gene. We did not detect a second CRB2 sequence variant on the other allele in the LCA patients and isolated RP patient that carried the remaining five amino acid substitutions (p.P46L, p.V97L, p.P116L, p.E187D, p.A351T), although we may have missed large deletions or rearrangements. Nevertheless, this renders autosomal recessive inheritance of CRB2 sequence variants as a cause of RP and LCA highly unlikely, but does not rule out digenic or polygenic mechanisms, or a de novo introduction of a dominant mutation. Interestingly, polygenic inheritance has recently been proposed for LCA [51]. Sequence variant p.E187D affects a residue that is conserved in the cbEGF-like domains, and which may provide ligands for binding of the calcium ion. Introduction of the longer side chain may affect calcium binding and disrupt the domain structure [34]. It is noteworthy that the same substitution (p.E1073D) at a similar position in one the cbEGF-like domains of fibrillin-1 results in a connective tissue disease [52].
Although we cannot completely rule out pathogenicity of some CRB2 sequence variants, this study shows that they are not a common cause of autosomal recessive RP and LCA. This study suggests that the CRB2 gene is highly polymorphic in comparison to the CRB1 gene [45]. Similarly, a retina specific paralog of the RP1 gene (RP1L1) is also highly polymorphic, and no RP1L1 mutations were detected in patients with retinal dystrophies [53,54]. It is possible that loss or altered function of CRB2 in humans is associated with a more complex clinical phenotype due to its expression in kidney. CRB2 therefore represents a candidate gene for cerebello-oculo-renal syndromes, such as Joubert syndrome, Dekaba-Arima syndrome, Senior-Löken syndrome, and COACH syndrome. A locus for Joubert syndrome maps to 9q34.3 [55], but CRB2 does not reside in the critical interval.
Acknowledgements
We thank G. C. F. van Duijnhoven, S. D. van der Velde-Visser, and Y. J. M. de Kok for excellent technical assistance. This work was supported by grants from the Foundation Fighting Blindness USA, N-CB-0600-0003 (FPMC and AIdH), the European Community, QLG3-CT-2002-01266 (FPMC and PR), and by kind donations of the Algemene Vereniging ter Voorkoming van Blindheid, the Gelderse Blindenvereniging, the Rotterdamse Vereniging Blindenbelangen, the Stichting Blindenhulp, the Stichting OOG, and the Stichting voor Ooglijders (JAJMvdH, FPMC, and CBH).
References
1. den Hollander AI, ten Brink JB, de Kok YJ, van Soest S, van den
Born LI, van Driel MA, van de Pol DJ, Payne AM, Bhattacharya SS, Kellner
U, Hoyng CB, Westerveld A, Brunner HG, Bleeker-Wagemakers EM, Deutman
AF, Heckenlively JR, Cremers FP, Bergen AA. Mutations in a human
homologue of Drosophila crumbs cause retinitis pigmentosa (RP12). Nat
Genet 1999; 23:217-21.
2. Bernal S, Calaf M, Garcia-Hoyos M, Garcia-Sandoval B, Rosell J,
Adan A, Ayuso C, Baiget M. Study of the involvement of the RGR, CRPB1,
and CRB1 genes in the pathogenesis of autosomal recessive retinitis
pigmentosa. J Med Genet 2003; 40:e89.
3. Khaliq S, Abid A, Hameed A, Anwar K, Mohyuddin A, Azmat Z, Shami
SA, Ismail M, Mehdi SQ. Mutation screening of Pakistani families with
congenital eye disorders. Exp Eye Res 2003; 76:343-8.
4. Lotery AJ, Malik A, Shami SA, Sindhi M, Chohan B, Maqbool C, Moore
PA, Denton MJ, Stone EM. CRB1 mutations may result in retinitis
pigmentosa without para-arteriolar RPE preservation. Ophthalmic Genet
2001; 22:163-9.
5. den Hollander AI, Heckenlively JR, van den Born LI, de Kok YJ, van
der Velde-Visser SD, Kellner U, Jurklies B, van Schooneveld MJ,
Blankenagel A, Rohrschneider K, Wissinger B, Cruysberg JR, Deutman AF,
Brunner HG, Apfelstedt-Sylla E, Hoyng CB, Cremers FP. Leber congenital
amaurosis and retinitis pigmentosa with Coats-like exudative
vasculopathy are associated with mutations in the crumbs homologue 1
(CRB1) gene. Am J Hum Genet 2001; 69:198-203. Erratum in: Am J Hum Genet
2001; 69:1160.
6. Lotery AJ, Jacobson SG, Fishman GA, Weleber RG, Fulton AB,
Namperumalsamy P, Heon E, Levin AV, Grover S, Rosenow JR, Kopp KK,
Sheffield VC, Stone EM. Mutations in the CRB1 gene cause Leber
congenital amaurosis. Arch Ophthalmol 2001; 119:415-20.
7. Hanein S, Perrault I, Gerber S, Tanguy G, Barbet F, Ducroq D,
Calvas P, Dollfus H, Hamel C, Lopponen T, Munier F, Santos L, Shalev S,
Zafeiriou D, Dufier JL, Munnich A, Rozet JM, Kaplan J. Leber congenital
amaurosis: comprehensive survey of the genetic heterogeneity, refinement
of the clinical definition, and genotype-phenotype correlations as a
strategy for molecular diagnosis. Hum Mutat 2004; 23:306-17.
8. Tepass U, Theres C, Knust E. crumbs encodes an EGF-like protein
expressed on apical membranes of Drosophila epithelial cells and
required for organization of epithelia. Cell 1990; 61:787-99.
9. Wodarz A, Hinz U, Engelbert M, Knust E. Expression of crumbs
confers apical character on plasma membrane domains of ectodermal
epithelia of Drosophila. Cell 1995; 82:67-76.
10. Pellikka M, Tanentzapf G, Pinto M, Smith C, McGlade CJ, Ready DF,
Tepass U. Crumbs, the Drosophila homologue of human CRB1/RP12, is
essential for photoreceptor morphogenesis. Nature 2002; 416:143-9.
11. Izaddoost S, Nam SC, Bhat MA, Bellen HJ, Choi KW. Drosophila
Crumbs is a positional cue in photoreceptor adherens junctions and
rhabdomeres. Nature 2002; 416:178-83.
12. Lemmers C, Medina E, Delgrossi MH, Michel D, Arsanto JP, Le Bivic
A. hINADl/PATJ, a homolog of discs lost, interacts with crumbs and
localizes to tight junctions in human epithelial cells. J Biol Chem
2002; 277:25408-15.
13. Makarova O, Roh MH, Liu CJ, Laurinec S, Margolis B. Mammalian
Crumbs3 is a small transmembrane protein linked to protein associated
with Lin-7 (Pals1). Gene 2003; 302:21-9.
14. Robinson PN, Godfrey M. The molecular genetics of Marfan syndrome
and related microfibrillopathies. J Med Genet 2000; 37:9-25.
15. Stone EM, Braun TA, Russell SR, Kuehn MH, Lotery AJ, Moore PA,
Eastman CG, Casavant TL, Sheffield VC. Missense variations in the
fibulin 5 gene and age-related macular degeneration. N Engl J Med 2004;
351:346-53.
16. Schultz DW, Klein ML, Humpert AJ, Luzier CW, Persun V, Schain M,
Mahan A, Runckel C, Cassera M, Vittal V, Doyle TM, Martin TM, Weleber
RG, Francis PJ, Acott TS. Analysis of the ARMD1 locus: evidence that a
mutation in HEMICENTIN-1 is associated with age-related macular
degeneration in a large family. Hum Mol Genet 2003; 12:3315-23.
17. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W,
Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein
database search programs. Nucleic Acids Res 1997; 25:3389-402.
18. Karolchik D, Baertsch R, Diekhans M, Furey TS, Hinrichs A, Lu YT,
Roskin KM, Schwartz M, Sugnet CW, Thomas DJ, Weber RJ, Haussler D, Kent
WJ, University of California Santa Cruz. The UCSC Genome Browser
Database. Nucleic Acids Res 2003; 31:51-4.
19. Letunic I, Goodstadt L, Dickens NJ, Doerks T, Schultz J, Mott R,
Ciccarelli F, Copley RR, Ponting CP, Bork P. Recent improvements to the
SMART domain-based sequence annotation resource. Nucleic Acids Res 2002;
30:242-4.
20. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the
sensitivity of progressive multiple sequence alignment through sequence
weighting, position-specific gap penalties and weight matrix choice.
Nucleic Acids Res 1994; 22:4673-80.
21. Dunn KC, Aotaki-Keen AE, Putkey FR, Hjelmeland LM. ARPE-19, a
human retinal pigment epithelial cell line with differentiated
properties. Exp Eye Res 1996; 62:155-69.
22. Luijendijk MW, van de Pol TJ, van Duijnhoven G, den Hollander AI,
ten Caat J, van Limpt V, Brunner HG, Kremer H, Cremers FP. Cloning,
characterization, and mRNA expression analysis of novel human fetal
cochlear cDNAs. Genomics 2003; 82:480-90.
23. den Hollander AI, van Driel MA, de Kok YJ, van de Pol DJ, Hoyng
CB, Brunner HG, Deutman AF, Cremers FP. Isolation and mapping of novel
candidate genes for retinal disorders using suppression subtractive
hybridization. Genomics 1999; 58:240-9.
24. Hall J, Nanthakumar E. Automated fluorescent genoytping. In: Dracopoli NC, Haines JL, Korf BR, Morton CC, Seidman CE, Seidman JG, Smith DR, editors. Current protocols in human genetics. John Wiley & Sons; New York; 2000. p. 2.8.1-19.
25. Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C,
Kalsheker N, Smith JC, Markham AF. Analysis of any point mutation in
DNA. The amplification refractory mutation system (ARMS). Nucleic Acids
Res 1989; 17:2503-16.
26. Little S. Amplification-refractory mutation system (ARMS) analysis of point mutations. In: Dracopoli NC, Haines JL, Korf BR, Morton CC, Seidman CE, Seidman JG, Smith DR, editors. Current protocols in human genetics. New York: John Wiley & Sons; 2000. p. 9.8.1-12.
27. Rebay I, Fleming RJ, Fehon RG, Cherbas L, Cherbas P,
Artavanis-Tsakonas S. Specific EGF repeats of Notch mediate interactions
with Delta and Serrate: implications for Notch as a multifunctional
receptor. Cell 1991; 67:687-99.
28. Yurchenco PD, Sung U, Ward MD, Yamada Y, O'Rear JJ. Recombinant
laminin G domain mediates myoblast adhesion and heparin binding. J Biol
Chem 1993; 268:8356-65.
29. Reinhardt DP, Sasaki T, Dzamba BJ, Keene DR, Chu ML, Gohring W,
Timpl R, Sakai LY. Fibrillin-1 and fibulin-2 interact and are
colocalized in some tissues. J Biol Chem 1996; 271:19489-96.
30. Adam S, Gohring W, Wiedemann H, Chu ML, Timpl R, Kostka G.
Binding of fibulin-1 to nidogen depends on its C-terminal globular
domain and a specific array of calcium-binding epidermal growth
factor-like (EG) modules. J Mol Biol 1997; 272:226-36.
31. Beckmann G, Hanke J, Bork P, Reich JG. Merging extracellular
domains: fold prediction for laminin G-like and amino-terminal
thrombospondin-like modules based on homology to pentraxins. J Mol Biol
1998; 275:725-30.
32. Cooke RM, Wilkinson AJ, Baron M, Pastore A, Tappin MJ, Campbell
ID, Gregory H, Sheard B. The solution structure of human epidermal
growth factor. Nature 1987; 327:339-41.
33. Handford PA, Mayhew M, Baron M, Winship PR, Campbell ID, Brownlee
GG. Key residues involved in calcium-binding motifs in EGF-like domains.
Nature 1991; 351:164-7.
34. Downing AK, Knott V, Werner JM, Cardy CM, Campbell ID, Handford
PA. Solution structure of a pair of calcium-binding epidermal growth
factor-like domains: implications for the Marfan syndrome and other
genetic disorders. Cell 1996; 85:597-605.
35. Bhat MA, Izaddoost S, Lu Y, Cho KO, Choi KW, Bellen HJ. Discs
Lost, a novel multi-PDZ domain protein, establishes and maintains
epithelial polarity. Cell 1999; 96:833-45. Erratum in: Cell. 2003;
115:765-6.
36. Klebes A, Knust E. A conserved motif in Crumbs is required for
E-cadherin localisation and zonula adherens formation in Drosophila.
Curr Biol 2000; 10:76-85.
37. Bachmann A, Schneider M, Theilenberg E, Grawe F, Knust E.
Drosophila Stardust is a partner of Crumbs in the control of epithelial
cell polarity. Nature 2001; 414:638-43.
38. Hong Y, Stronach B, Perrimon N, Jan LY, Jan YN. Drosophila
Stardust interacts with Crumbs to control polarity of epithelia but not
neuroblasts. Nature 2001; 414:634-8.
39. Medina E, Williams J, Klipfell E, Zarnescu D, Thomas G, Le Bivic
A. Crumbs interacts with moesin and beta(Heavy)-spectrin in the apical
membrane skeleton of Drosophila. J Cell Biol 2002; 158:941-51.
40. Roh MH, Makarova O, Liu CJ, Shin K, Lee S, Laurinec S, Goyal M,
Wiggins R, Margolis B. The Maguk protein, Pals1, functions as an
adapter, linking mammalian homologues of Crumbs and Discs Lost. J Cell
Biol 2002; 157:161-72.
41. Roh MH, Fan S, Liu CJ, Margolis B. The Crumbs3-Pals1 complex
participates in the establishment of polarity in mammalian epithelial
cells. J Cell Sci 2003; 116:2895-906.
42. Lemmers C, Michel D, Lane-Guermonprez L, Delgrossi MH, Medina E,
Arsanto JP, Le Bivic A. CRB3 binds directly to Par6 and regulates the
morphogenesis of the tight junctions in mammalian epithelial cells. Mol
Biol Cell 2004; 15:1324-33.
43. Livingstone CD, Barton GJ. Protein sequence alignments: a
strategy for the hierarchical analysis of residue conservation. Comput
Appl Biosci 1993; 9:745-56.
44. Henikoff S, Henikoff JG. Amino acid substitution matrices from
protein blocks. Proc Natl Acad Sci U S A 1992; 89:10915-9.
45. den Hollander AI, Davis J, van der Velde-Visser SD, Zonneveld MN,
Pierrottet CO, Koenekoop RK, Kellner U, van den Born LI, Heckenlively
JR, Hoyng CB, Handford PA, Roepman R, Cremers FP. CRB1 mutation spectrum
in inherited retinal dystrophies. Hum Mutat 2004; 24:355-69.
46. den Hollander AI, Johnson K, de Kok YJ, Klebes A, Brunner HG,
Knust E, Cremers FP. CRB1 has a cytoplasmic domain that is functionally
conserved between human and Drosophila. Hum Mol Genet 2001; 10:2767-73.
47. Katoh M, Katoh M. Identification and characterization of Crumbs
homolog 2 gene at human chromosome 9q33.3. Int J Oncol 2004; 24:743-9.
48. van de Pavert SA, Kantardzhieva A, Malysheva A, Meuleman J,
Versteeg I, Levelt C, Klooster J, Geiger S, Seeliger MW, Rashbass P, Le
Bivic A, Wijnholds J. Crumbs homologue 1 is required for maintenance of
photoreceptor cell polarization and adhesion during light exposure. J
Cell Sci 2004; 117:4169-77.
49. den Hollander AI, Ghiani M, de Kok YJ, Wijnholds J, Ballabio A,
Cremers FP, Broccoli V. Isolation of Crb1, a mouse homologue of
Drosophila crumbs, and analysis of its expression pattern in eye and
brain. Mech Dev 2002; 110:203-7.
50. Watanabe T, Miyatani S, Katoh I, Kobayashi S, Ikawa Y. Expression
of a novel secretory form (Crb1s) of mouse Crumbs homologue Crb1 in skin
development. Biochem Biophys Res Commun 2004; 313:263-70.
51. Allikmets RL, Zernant J, Perrault I, den Hollander A, Dharmaraj S, Cremers FPM, Kaplan J, Koenekoop RK, Maumenee I. Multiallelic inheritance and/or modifier alleles in Leber congenital amaurosis: analysis with the LCA disease chip. ARVO Annual Meeting; 2004 April 25-29; Fort Lauderdale (FL).
52. Wang M, Kishnani P, Decker-Phillips M, Kahler SG, Chen YT,
Godfrey M. Double mutant fibrillin-1 (FBN1) allele in a patient with
neonatal Marfan syndrome. J Med Genet 1996; 33:760-3.
53. Bowne SJ, Daiger SP, Malone KA, Heckenlively JR, Kennan A,
Humphries P, Hughbanks-Wheaton D, Birch DG, Liu Q, Pierce EA, Zuo J,
Huang Q, Donovan DD, Sullivan LS. Characterization of RP1L1, a highly
polymorphic paralog of the retinitis pigmentosa 1 (RP1) gene. Mol Vis
2003; 9:129-37 <http://www.molvis.org/molvis/v9/a19/>.
54. Conte I, Lestingi M, den Hollander A, Alfano G, Ziviello C,
Pugliese M, Circolo D, Caccioppoli C, Ciccodicola A, Banfi S.
Identification and characterisation of the retinitis pigmentosa 1-like1
gene (RP1L1): a novel candidate for retinal degenerations. Eur J Hum
Genet 2003; 11:155-62.
55. Saar K, Al-Gazali L, Sztriha L, Rueschendorf F, Nur-E-Kamal M,
Reis A, Bayoumi R. Homozygosity mapping in families with Joubert
syndrome identifies a locus on chromosome 9q34.3 and evidence for
genetic heterogeneity. Am J Hum Genet 1999; 65:1666-71.