CEP290, a with many faces: mutation overview and presentation of CEP290base Frauke Coppieters, Steve Lefever, Bart P. Leroy, Elfride Bw de Baere

To cite this version:

Frauke Coppieters, Steve Lefever, Bart P. Leroy, Elfride Bw de Baere. CEP290, a gene with many faces: mutation overview and presentation of CEP290base. Human Mutation, Wiley, 2010, 31 (10), pp.1097. ￿10.1002/humu.21337￿. ￿hal-00563445￿

HAL Id: hal-00563445 https://hal.archives-ouvertes.fr/hal-00563445 Submitted on 5 Feb 2011

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Human Mutation

CEP290 , a gene with many faces: mutation overview and presentation of CEP290 base

For Peer Review

Journal: Human Mutation

Manuscript ID: humu-2010-0040.R1

Wiley - Manuscript type: Mutation Update

Date Submitted by the 18-Jun-2010 Author:

Complete List of Authors: Coppieters, Frauke; Ghent University Hospital, Center for Medical Genetics Lefever, Steve; Ghent University Hospital, Center for Medical Genetics Leroy, Bart; Ghent University Hospital, Department of Ophthalmology; Ghent University Hospital, Center for Medical Genetics De Baere, Elfride; Ghent University Hospital, Center for Medical Genetics

CEP290, -specific database, genotype-phenotype correlations, Key Words: modifiers, ciliary proteome

John Wiley & Sons, Inc. Page 1 of 51 Human Mutation

1 2 CEP290 , a gene with many faces: mutation overview and presentation of 3 4 5 CEP290 base 6 7 8 9 Deleted : Jo Vandesompele 1, 10 11 Frauke Coppieters 1*, Steve Lefever 1*, Bart P. Leroy 1,2 , Elfride De Baere 1 12 13 14 15 16 1Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium 17 18 For Peer Review 19 2Department of Ophthalmology, Ghent University Hospital, Ghent, Belgium 20

21 * 22 These two authors contributed equally to this work. 23 24 25 26 27 Address for Correspondence: Elfride De Baere, MD, PhD, [email protected], Center 28 29 for Medical Genetics, Ghent University Hospital, De Pintelaan 185, B- 9000 Ghent, Belgium, 30 31 Phone: 32-9-332.5186 Fax: 32-9-332.6549. 32 33 34 35 36

37 38 39 40 41 42 43 44

45 46 47 48 49 1 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 2 of 51

1 2 Abstract 3 4 5 6 are an emerging group of disorders, caused by mutations in ciliary . One of the Deleted : 7 of most intriguing disease genes associated with ciliopathies is CEP290 , mutations in which cause a 8 9 wide variety of distinct phenotypes, ranging from isolated blindness over Senior-Loken syndrome 10 11 (SLS), (NPHP), (related disorders) (JS[RD]), Bardet-Biedl 12 13 syndrome (BBS) to the lethal Meckel-Grüber syndrome (MKS). Despite the identification of over 14 15 100 unique CEP290 mutations, no clear genotype-phenotype correlations could yet be 16 17 established and consequently the predictive power of a CEP290 -related genotype remains 18 For Peer Review 19 limited. One of the challenges is a better understanding of second-site modifiers. In this respect , 20 Deleted : n 21 there is a growing interest in the potential modifying effects of variations in genes encoding other Deleted : emerging 22 Deleted : other components of the 23 members of the ciliary proteome which interact with CEP290 . ciliary proteome, possibly modifying 24 phenotypes caused by mutations in 25 interacting Deleted : 26 Here, we provide an overview of all CEP290 mutations identified so far, with their associated 27 Deleted : For this purpose 28 phenotypes. To this end , we developed CEP290 base , a locus-specific mutation database that links 29 30 mutations with patients and their phenotypes (medgen.ugent.be/cep290base) . 31 32 33 Key words : CEP290 , locus-specific database, genotype-phenotype correlations, modifiers, ciliary 34 35 proteome 36 37 38 39 40 41 42 43 44 45

46 47 48 49 2 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 3 of 51 Human Mutation

1 2 Background 3 4 5 6 Cilia are highly conserved organelles that are essential for many cell types. Apart from their Deleted : 7 exert obvious role in motility and transport of fluids and particles over epithelial surfaces, they have 8 Deleted : reception and 9 numerous other functions such as signal transduction (Berbari et al., 2009). The extensive 10 11 presence of cilia throughout the whole body might explain the wide range of phenotypes 12 13 associated with mutations in genes encoding ciliary proteins (Gerdes et al., 2009; Nigg and Raff, 14 15 2009). 16 17 18 One of the most intriguing diseaseFor genes associated Peer with ciliopathies Review is CEP290 (MIM[610142]) , 19 20 as the phenotypic spectrum of its mutations ranges from isolated blindness to the lethal Meckel- 21 22 Grüber syndrome (MKS). The gene was initially identified as disease gene for Joubert syndrome 23 24 (related disorders) (JS[RD]) and Senior-Loken syndrome (SLS) (Sayer et al., 2006; Valente et al., 25 26 2006). Within a few years, Leber Congenital Amaurosis (LCA), MKS and Bardet-Biedl 27 28 syndrome (BBS) expanded the list of partially overlapping yet distinct disorders caused by 29 30 CEP290 mutations (den Hollander et al., 2006; Baala et al., 2007a; Leitch et al., 2008). Although 31 32 these are essentially autosomal recessive (AR) monogenic diseases, epistatic effects of modifier 33 Deleted : other 34 alleles in additional ciliary genes should not be underestimated in the development of their 35 36 phenotypes. 37 38 39 The first clone corresponding with CEP290 , KIAA0373 , was identified through sequencing of 40 41 100 new cDNA clones from cDNA libraries (Nagase et al., 1997). Three years later, 42 43 Chen and Shou independently cloned CEP290 as 3H11Ag, encoding an antigen for the 44 45 monoclonal antibody 3H11 which specifically recognizes cancer cells from various tissues (Chen 46 47 and Shou, 2001). It was predicted that about 60% of the residues form coiled-coil (CC) structures 48 49 3 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 4 of 51

1 2 and that the has four potential dimeric CC regions. In addition, the protein displays high 3 4 similarity to myosin among different species and was predicted to have a partial structural 5 Deleted : and a myosin -like domain 6 maintenance of (SMC) conserved domain. Several predicted motifs suggested 7 8 potential modifications such as N-glycosylation, tyrosine sulfation and phosphorylation (Guo et 9 10 al., 2004). In 2003, Andersen and colleagues detected the KIAA0373 gene product in human 11 12 following mass-spectrometry-based proteomic analysis. The protein was called 13 14 Cep290 according to its centrosomal location and approximate relative molecular mass, and was 15 16 predicted to contain nine CCs (Andersen et al., 2003). Upon the identification of CEP290 as a 17 Deleted : the 18 novel disease gene for JS, CEP290For was further Peer characterized. ReviewAnalysis of the deduced amino acid Deleted : protein 19 Deleted : CEP290 20 (AA) sequence revealed 13 putative CC domains, a region with homology to SMC chromosomal 21 22 segregation , a bipartite nuclear localization signal, six RepA/Rep+ protein KID motifs, 23 24 three tropomyosin homology domains and an ATP/GTP binding site motif A (Sayer et al., 2006). 25 26 The CEP290 gene as currently annotated, spans 54 exons with the coding region starting in exon 27 28 2 (NM_025114.3). Given the broad allelic spectrum, the complexity of associated phenotypes 29 30 and the presumed influence of modifier genes, the establishment of genotype-phenotype 31 32 correlations poses a major challenge. 33 34 35 Variants in CEP290 36 37 38 Mutations 39 Deleted : 11 1 40 So far, 112 distinct mutations have been identified ( Figure 1, Supp. Table S1, NM_025114.3 ). Deleted : Table 1 41 Deleted : 39 42 The vast majority of CEP290 mutations are truncating, with 40 nonsense and 48 frameshift 43 44 mutations reported so far. All frameshifts are caused by small deletions or insertions, with the 45 46 exception of two indels (c.381_382delinsT and c.5865_5867delinsGG). Three deletions and one 47 48 duplication directly lead to a premature termination codon (PTC) without the incorporation of 49 4 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 5 of 51 Human Mutation

1 Deleted : , 2 novel AAs (c.1550del, c.2906dup, c.3175del and c.5046del). Taking into account the position of Deleted : and c.2906dup 3 Deleted : -most 4 the PTC relative to the ultimate 3′ exon–exon junction, 85 truncating mutations are assumed to Deleted : 84 5 Deleted : be 6 undergo nonsense mediated decay (NMD) (Nagy and Maquat, 1998). However, the p.Arg151X 7 8 mutation was shown to result in alternative transcripts, lacking exon 7 or both exon 7 and 8, 9 Deleted : In contrast, three 10 leaving the open reading frame intact (Littink et al. , 2010 ). Three truncating mutations located in 11 Deleted : to 12 the last exon are expected to escape NMD and render a protein product (c.7318_7321dup, 13 14 c.7341dup and c.7366_7369del). 15 16 Deleted : 87 17 In addition to the 88 truncating mutations caused by small variations, a large heterozygous 18 Deleted : following copy number For Peer Review profiling of the CEP290 gene in nine 19 deletion was recently identified in a patient with cerebello-oculo-renal syndrome (CORS). The patients carrying a single CEP290 20 mutated allele (4 CORS, 1 MKS and 4 LCA) 21 deleted region spanned 76,844 bp at the genomic level, encompassing the last 13 coding exons of 22 23 CEP290 , the entire C12orf29 gene and part of the C12orf50 gene 24 25 (CEP290:c.5709+2352_54_C12orf50:c.290–1375_77del) (Travaglini et al., 2009). 26 Deleted : three 27 The remaining 23 mutations comprise 3 missense mutations and 20 mutations predicted to 28 Deleted : ( Table 1 ) 29 influence splicing. Of the latter, 12 mutations affect consensus donor or acceptor splice sites, 30 31 whereas three mutations are located within 20 nucleotides surrounding the exon. The first two, 32 33 c.103-13_103-18del and c.6271-8T>G, were considered to be likely pathogenic because they 34 35 decreased the scores of the normal splice sites and because they are absent in more than 115 36 Deleted : reported 37 control individuals (Tory et al., 2007). The third one, c.1711+5A>G, was presumed to result in Deleted : probably 38 39 both abnormally and normally spliced transcripts (Perrault et al., 2007). Splice site prediction 40 41 scores, however, remain unchanged for this variant (Alamut v.1.5, data not shown). In addition, 42 43 an aberrant splicing pattern was predicted for the c.1824G>A mutation affecting the last 44 45 nucleotide of exon 18 (Coppieters et al., 2010). Surprisingly, the most recurrent mutation, 46 47 c.2991+1655A>G, represents a deep intronic mutation. It creates a strong splice donor site that 48 49 5 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 6 of 51

1 2 results in the insertion of a 128-bp cryptic exon between exons 26 and 27, thereby leading to a 3 4 PTC immediately downstream of exon 26 (p.Cys998X) (den Hollander et al., 2006). Apart from 5 6 these 17 substitutions, two deletions and one indel overspan an intron-exon boundary (c.2218- 7 8 4_2222del, c.2218-15_2220del and c.3310-1_3310delinsAA). 9 10 11 So far, only three missense variants have been described with a probable pathogenic effect (see 12 13 “Polymorphisms and unclassified variants” for other missense variants). Two affect the start 14 Deleted : at the N-terminus of the protein. This change 15 codon (c.1A>G and c.2T>A) whereas the third, p.Trp7Cys, affects a highly conserved AA and is 16 Deleted : affect 17 predicted to disrupt protein function according to SIFT, PolyPhen and Grantham matrix . 18 For Peer Review Deleted : , but 19 However, the mutant protein was correctly localized at centrosomes in mouse inner medullary Deleted : murine 20 Deleted : than 21 collecting duct (IMCD-3) cells, suggesting a pathogenic mechanism different from 22 23 mislocalization (Valente et al., 2006). 24 25 Interestingly, some mutations cluster in the same region or even codon, suggesting the existence 26 27 of mutation hotspots. In nine coding regions, two or more mutations arose either with a different 28 Deleted : _382delinsT, c.384_385del and c.384_387del 29 effect on protein level (start codon; c.381 -387 ; c.1859 -1862; c.3175 -3176; c.4965 -4966 ; c.5515 - Deleted : 30 _ Deleted : del and c.1860_1861del 31 5537; c.5865 -5867; c.6869 -6870) or with an identical predicted protein effect (c.4114 -4116; 32 Deleted : dup, c.3175del and c. Deleted : 33 c.4962 -4966). In addition, one donor and three acceptor splice sites displayed more than one del 34 Deleted : _4966del and c.4966G>T 35 mutation (c.180+1G>T and c.180+2T>A; c.2218-2A>C, c.2218-4_2222del and c.2218- Deleted : _5518del and c.5519_ 36 Deleted : del 37 15_2220del; c.3104-1G>A and c.3104-2A>G; c.3310-1_3310delinsAA and c.3310-1G>C). In Deleted : _ 38 Deleted : delinsGG and c.586 6G>T 39 general, mutations are scattered throughout the protein, with some clustering in CCs III, XI and Deleted : del and c. 40 Deleted : 41 del XII (Figure 1) . Coiled-coils might be involved in the overall conformation of CEP290. Therefore, Deleted : 42 _4115del and c.4115_ Deleted : del 43 mutations in them might affect the accessibility of interacting proteins (Schafer et al., 2008). 44 Deleted : _4963d el and c.4965_ 45 Deleted : del 46 Deleted : Mutations located in the SMC1-like domain might influence 47 intraflagellar protein trafficking (Chang 48 et al., 2006). 49 6 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 7 of 51 Human Mutation

1 Deleted : 79 2 A total of 83 mutations are unique whereas 26 mutations have been reported in less than 10 Deleted : 29 3 Deleted : Table 1 4 families ( Supp. Table S1 ). A few other mutations occurred multiple times and might even 5 Deleted : mutation 6 represent founder mutations. The most recurrent one is c.2991+1655A>G. In northwestern 7 8 Europe, this mutation occurs in up to 26% of all LCA cases (den Hollander et al., 2006; Perrault 9 10 et al., 2007; Coppieters et al., 2010). In Southern Europe, Korea, Southern India and Saudi- 11 Deleted : this mutation 12 Arabia, however, c.2991+1655A>G has a significantly lower prevalence (Simonelli et al., 2007; Deleted : been described to have 13 Deleted : in LCA 14 Vallespin et al., 2007; Seong et al., 2008; Li et al., 2009; Sundaresan et al., 2009). The second- Deleted : Contrary to the third-most 15 frequent mutation p.Gly1890X that was 16 most frequent mutation, p.Lys1575X, was so far only reported in probands originating from found in 11 families worldwide, t 17 Deleted : North 18 France (Lille) and northern BelgiumFor (Brancati Peer et al., 2007; Perrault Review et al., 2007; Coppieters et al., 19 Deleted : of the latter 20 2010). Haplotype analysis in seven non-consanguineous families revealed a common intragenic 21 22 allele but distinct extragenic haplotypes, suggesting an ancient mutation (Perrault et al., 2007). 23 24 For p.Ala1832ProfsX19, allele sharing analysis in two families with MKS originating from 25 26 Kosovo and Kosovo-Albania was in favor of a common founder haplotype encompassing 27 28 approximately 3 Mb (Frank et al., 2008). In contrast, different haplotypes in two MKS families of 29 Deleted : recurrent 30 Tunisian and French origin point to a mutation hotspot for p.Asp128GlufsX34 (Baala et al., Deleted : rather than a founder effect 31 32 2007a). All mutations segregated in the parents (as far as this could be assessed), with the 33 34 exception of p.Gln2111X, which was reported to arise de novo in a patient with CORS (Sayer et 35 36 al., 2006). 37 Deleted : 38 could be In 18 patients, only one mutation was identified. This might be due to an inability of standard 39 40 PCR-based techniques to identify mutations such as deep intronic variants, large genomic 41 42 rearrangements or regulatory mutations located in promoter or enhancer/silencer elements. On 43 44 the other hand, the heterozygous CEP290 mutation might represent a modifying allele, 45 Deleted : recessive 46 potentially influencing the clinical expression of two AR mutations in another ciliary gene. 47 48 49 7 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 8 of 51

1 Deleted : These 2 Similar arguments could also apply for five families with isolated NPHP in which no mutations 3 4 could be identified at all, despite linkage to a region containing CEP290 (Helou et al., 2007). 5 Deleted : Again 6 Alternatively , another as yet unidentified NPHP gene could segregate in linkage disequilibrium 7 8 with CEP290 in these families. 9 10 11 Polymorphisms and unclassified variants 12 Deleted : in 13 Several polymorphisms have been described in CEP290 , for which we refer to dbSNP 14 15 (http://www.ncbi.nlm.nih.gov/projects/SNP) and Brancati and colleagues (Brancati et al., 2007). 16 17 They were predicted not to alter splicing patterns or to impair protein function, and/or were 18 For Peer Review 19 present in over 2% of parents of affected individuals (Brancati et al., 2007). In addition, the 20 Deleted : Table 2 21 pathogenic potential of some variants is indefinite ( Supp. Table S2 ). Indeed, for several missense Deleted : F 22 23 variants, in silico predictions are not conclusive (PolyPhen, SIFT and Grantham matrix). The first 24 25 one is p.Asp664Gly. Despite the identification of this variant in a heterozygous state in a patient 26 27 with JS and renal involvement, p.Asp664Gly also occurred in a healthy parent of another family, 28 29 but not in his affected child (Brancati et al., 2007; Helou et al., 2007). Interestingly, Tory and Deleted : 30 four 31 colleagues screened this variant together with three other CEP290 missense variants 32 33 (p.Glu277Gln, p.Lys838Glu and p.Arg1746Gln) in the context of modifier identification in the 34 35 following three cohorts: 1) 13 patients carrying NPHP1 mutation(s) with neurological symptoms; 36 37 2) 77-82 patients carrying NPHP1 mutation(s) without neurological symptoms and 3) 132-154 38 healthy control subjects. No significant difference in frequencies was found among these three 39 40 cohorts, however (Tory et al., 2007). Other missense variants of unknown significance are 41 42 p.Leu906Trp, p.Asn2228Lys, p.Ala1566Pro and p.Leu1694Pro (Brancati et al., 2007; Coppieters 43 44 et al., 2010). The latter two were found in two LCA patients, in compound heterozygosity with a 45 46 nonsense and splice site mutation, respectively, and segregated in healthy parents (Coppieters et 47 48 49 8 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 9 of 51 Human Mutation

1 2 al., 2010). Two additional unclassified variants include in-frame deletions. A p.Glu1554del 3 4 variant segregated with p.Phe1950LeufsX15 in an LCA patient (Perrault et al., 2007), while a 5 6 heterozygous p.Lys2437del variant was identified in a patient with isolated NPHP originating Deleted : Table 2 7 8 from consanguineous parents (Helou et al., 2007) ( Supp. Table S2 ). 9 10 11 Mutation database 12 13 14 To provide a clear overview of all mutations and variants identified so far in CEP290 , we 15 Deleted : the locus-specific mutation 16 developed CEP290 base (medgen.ugent.be/cep290base). This locus-specific mutation database database 17 Deleted : is 18 uses a novel scheme in whichFor both genomic Peer and phenotypic Review data are available, providing the 19 20 possibility to link patients and their phenotypes to detailed variant information, and vice versa. 21 22 Information on variants can be retrieved using the Overview page or the Mutation Browser. 23 24 25 The Overview page displays all unique variants following HGVS guidelines 26 27 (http://www.hgvs.org/mutnomen) (NM_025114.3), with their occurrence and links to dbSNP, 28 29 UniProt (http://www.uniprot.org) and OMIM 30 31 (http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim). In addition, a scaled graphical 32 Deleted : o 33 representation of variants in the CEP290 protein is shown. Variants in the Overview or image are 34 35 linked to their variant-specific page which, if available, includes the protein domain (Sayer et al., 36 37 2006), as well as the frequency in control individuals, the RNA effect and the estimate of 38 39 pathogenic probability (Stone, 2003). For truncating mutations, it is indicated whether mRNA 40 41 might be subjected to NMD (Nagy and Maquat, 1998), and for missense mutations, the Grantham 42 43 score is provided (Grantham, 1974). The database also links to SIFT (http://sift.jcvi.org) and 44 45 PolyPhen (http://genetics.bwh.harvard.edu/pph) prediction servers, using the CEP290 GI number 46 47 (109255233) and UniProt ID (O15078) for automated input, respectively. Links to NetGene2 48 49 9 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 10 of 51

1 2 (http://www.cbs.dtu.dk/services/NetGene2) and the Berkeley Drosophila Genome Project 3 4 (http://www.fruitfly.org/seq_tools/splice.html) are available for splice site mutations. A list of 5 Deleted : 6 patients reported to carry the selected variant completes the variant -specific page. For each 7 Deleted : specific 8 patient, information regarding disease, gender, age, origin, segregation and parental 9 10 consanguinity is provided. Importantly, reported mutations are displayed using the nomenclature 11 Deleted : specific 12 of the original publication, allowing easy retrieval of the variant from literature. If available, 13 14 phenotypic information on ocular, renal, neurological and other signs appears by selecting the 15 16 patient’s ID. Disease calling was performed following the classification of Valente and 17 Deleted : who made a clear distinction 18 coworkers, and is based on theFor involvement Peer of different organ Review systems (Valente et al., 2008). Of between 19 Deleted : n 20 note, these phenotypes might be incomplete due to factors such as a clinical investigation in an Deleted : early 21 22 early disease stage . 23 24 Deleted : can be performed 25 The Mutation Browser enables custom querying. Both a quick and a more advanced search are 26 27 possible . The quick search uses a selection of variant nomenclatures or patient IDs to list all 28 29 patients carrying the selected variants, or all variants present in the selected patients. In the 30 31 advanced search, different parameters can be set within and between three sections: variant, 32 33 patient and source information. As in the quick search, the user has the choice between two 34 35 output options: a mutation or patient list. Finally, users can submit novel or known variants they 36 37 have identified.

38 Deleted : the novel database scheme of 39 In addition to the availability of phenotypic data in CEP290 base , it also includes variants in other Deleted : the database 40 41 genes that co-occur with CEP290 variants, thereby providing a unique opportunity to link 42 43 modifiers to associated clinical manifestations (see “Epistatic effect of other components of the 44 45 ciliary proteome”). The database is running on MySQL. PHP and JavaScript were used to Deleted : Mutation nomenclature is 46 based on the NCBI RefSeq NM_025114, 47 according to HGVS guidelines develop the web-based interface. (http://www.hgvs.org/mutnomen) . 48 49 10 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 11 of 51 Human Mutation

1 2 Biological relevance 3 4 5 6 Expression 7 The original KIAA0373 mRNA was found in kidney and ovary, and to a lesser extent in human 8 Deleted : human 9 thymus, prostate and testis tissue (Nagase et al., 1997). In addition, it was present in centrosomes 10 11 from a human lymphoblastoid cell line (KE-37) (Andersen et al., 2003). The independently 12 Deleted : expressed 13 cloned 3H11Ag mRNA was extensively distributed in embryonic tissue and in different human 14 15 cancerous tissues, but not in corresponding normal human tissues (Chen and Shou, 2001). The 16 17 150 C-terminal AAs of 3H11Ag appeared to be responsible for nuclear translocation. In addition, 18 For Peer Review 19 cytoplasmic presence was also established (Guo et al., 2004). Consistent with the localization of 20 21 the KIAA0373 protein, CEP290 was found at centrosomes of both ciliated and non-ciliated cells 22 23 (Chang et al., 2006; Sayer et al., 2006; Valente et al., 2006). This centrosomal localization of 24 25 CEP290 is dynamic throughout the cell cycle, with redistribution to the starting in 26 27 prometaphase (Sayer et al., 2006). During the interphase, two or four prominent spots were 28 29 observed in the G1 and G2 phase. In the G0 phase, CEP290 was found on both the daughter and 30 31 mother , the latter from which the primary is assembled (Tsang et al., 2008). 32 33 Although the centrosomal localization is - and -independent (Chang et al., 34 35 2006; Sayer et al., 2006), a major fraction of CEP290 is recruited to centriolar satellites along 36 37 polymerized (Kim et al., 2008). 38 39 40 In the rod-dominated mouse , CEP290 was detected in the connecting cilia, and to a lesser 41 42 extent in the inner segments (Chang et al., 2006; Sayer et al., 2006). Expression was also 43 44 established in primate cone photoreceptors (Cideciyan et al., 2007). In olfactory sensory neurons, 45 Deleted : is localized 46 CEP290 localizes to dendritic knobs (McEwen et al., 2007). A recent study on differential gene Deleted : Similar to 3H11Ag, CEP290 showed both cytoplasmic and nuclear 47 localization . 48 expression in the ventricular myocardium of newborn piglets identified CEP290 as being three- 49 11 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 12 of 51

1 Deleted : ¶ 2 fold more enriched in the right compared with the left ventricle (Torrado et al. , 2010 ). Knock- 3 Deleted : antisense 4 down experiments in zebrafish using morpholino s caused defects reminiscent of JS, comprising Deleted : oligonucleotides 5 Deleted : be ing 6 retinal, cerebellar and otic cavity developmental abnormalities as well as pronephric cyst 7 8 formation, ectopic brain tissue in the fourth ventricle and an abnormal mid-to-hindbrain region 9 10 associated with hydrocephalus (Sayer et al., 2006; Schafer et al., 2008). Alternatively spliced 11 12 transcripts/isoforms were identified by us in lymphocytes (data not shown) and observed as bands 13 14 of low molecular mass on immunoblot analysis in bovine retinal extracts (Chang et al., 2006). 15 16 17 Interaction with other (ciliary) proteins and function 18 For Peer Review Deleted : 19 Using a yeast two-hybrid screen and co-immunoprecipitation , Sayer and colleagues identified an Deleted : of a human fetal brain 20 expression library 21 interaction between the N-terminal third of CEP290 (exons 2-21) and the C-terminal two thirds Deleted : corresponding with 22 23 of the Activation Transcription Factor 4 (ATF4) protein. Moreover, CEP290 was able to activate 24 Deleted : can function both as a 25 ATF4-mediated transcription (Sayer et al., 2006). ATF4 has a protective function by regulating transcriptional activator and repressor. 26 The protein also plays Deleted : role 27 the adaptation of cells to metabolic and oxidative stress, and is required for skeletal and lens 28 Deleted : in Deleted : 29 development, and haematopoiesis (Ameri and Harris, 2008). In addition, association with G two components of the 30 γ13 olfactory G protein, 31 Deleted : , and G olf was identified in mouse olfactory epithelial tissue, suggesting a role of CEP290 in G 32 33 protein trafficking during olfactory perception (McEwen et al., 2007). 34 35 36 Moreover, CEP290 interacts with several centrosomal and ciliary proteins. Co- 37 38 immunoprecipitation experiments using mouse or bovine retinal extracts showed that CEP290 is 39 40 in complex with dynactin subunits p150 Glued and p50-dynamitin, subunit KIF3A, kinesin- 41 Deleted : PCM1, 42 associated protein (KAP3), the pericentriolar components γ- and pericentrin , the centriolar 43 Deleted : pericentrin, 44 marker centrin, PCM1, ninein, SMC1, SMC3, GTPase regulator 45 46 (RPGR ORF15 ) and RPGR-interacting protein 1 (RPGRIP1), but not with nucleophosmin, NPHP5 47 48 49 12 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 13 of 51 Human Mutation

1 2 or RP1 (Chang et al., 2006). In mouse olfactory epithelial tissue, association with p150 Glued , 3 4 KIF3A, RPGR ORF15 and γ-tubulin, but not BBS4 and IFT88, was also observed (McEwen et al., 5 Deleted : the pericentriolar components 6 2007). Of note, a subsequent study showed that pericentrin , γ-tubulin and centrin do not exactly Deleted : and 7 Deleted : , 8 co-localize with CEP290 (Kim et al., 2008). Deleted : the centriolar marker 9 10 11 Recently, a potential role for CEP290 in primary cilium assembly was established. Knock-down 12 13 of CEP290 using siRNAs in human retinal pigment epithelial cells caused a dramatic alteration in 14 15 the ability of cycling cells to assemble primary cilia on the one hand, and a disrupted migration of 16 17 mother to the cell cortex and primary cilia loss in quiescent cells on the other hand 18 For Peer Review 19 (Kim et al., 2008; Tsang et al., 2008). This observation might – at least partially - be attributed to 20 Deleted : At f 21 the association of CEP290 with two proteins. First, CEP290 was found to be recruited to Deleted : a major component of 22 centriolar satellites that 23 centriolar satellites by PCM-1, which is required for the organization of the cytoplasmic Deleted : and cooperates with the BBS 24 complex 25 microtubule network. Both depletion and overexpression of CEP290 results in redistribution of 26 27 PCM-1, thereby possibly influencing the transport function of PCM-1 granules between the 28 29 cytosol and (Kim et al., 2008). In addition, CEP290 recruits Rab8a, a small GTPase Deleted : in cooperation with a core 30 complex of BBS proteins 31 required for ciliary membrane elongation, at centrosomes and cilia (Kim et al., 2008; Tsang et al., 32 33 2008). The interaction with Rab8a requires the CEP290 AAs 1208-1695 (Tsang et al., 2008). In 34 35 growing cells (not yet capable of ciliogenesis), the latter function of CEP290 is probably 36 37 inhibited through an interaction with CP110, which prevents CEP290-dependent Rab8a 38 ciliogenesis (Tsang et al., 2008). Truncating CEP290 mutants defined the following amino- 39 40 terminal regions necessary for and sufficient to bind CP110: AAs 1-366, AAs 221-366 and AAs 41 42 362-822. The binding region of CP110 consists of AAs 1-223, with major involvement of AAs 43 44 67-82 (Tsang et al., 2008). 45 46 47 48 49 13 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 14 of 51

1 2 Interestingly, several other CEP290-interacting proteins are also associated with ciliopathies 3 Deleted : Table 3 4 (Table 1 ). A first one is RPGR. Although a yeast two-hybrid screen could not identify a direct 5 6 interaction with CEP290, co-immunoprecipitation assays suggested complex formation and both 7 Deleted : photoreceptors 8 proteins co-localized in IMCD-3 cells and dissociated mouse rod s (Chang et al., 2006). Mutations 9 10 in RPGR account for approximately 70-90% of X-linked retinitis pigmentosa (RP) and mainly 11 12 occur in isoforms containing the carboxy-terminal exon open reading frame 15 (RPGR ORF15 ), 13 14 which are highly expressed in connecting cilia of photoreceptors (Hong et al., 2003; Shu et al., 15 16 2007). In the rd16 mouse (see “Animal models”), mutant CEP290 binds RPGR ORF15 more avidly, 17 Deleted : subsequent 18 leading to aggregation of RPGRForORF15 in the Peer inner segments andReview redistribution of rhodopsin and 19 20 arrestin throughout the plasma membrane (Chang et al., 2006). 21 22 23 A second example of a CEP290-interacting protein also associated with ciliary disease is NPHP5 , Deleted : The NPHP5 gene was first 24 ORF15 described as a disease gene for SLS, 25 which is associated with SLS . The protein binds calmodulin and is in complex with RPGR correlating well with its expression in connecting cilia and outer segments (OS) 26 of photoreceptors and primary cilia of 27 (Otto et al., 2005). Despite the absence of a complex between CEP290 and NPHP5 in retinal renal epithelial cells. 28 Deleted : the N-terminal 29 extracts (Chang et al., 2006), NPHP5 was shown to specifically bind a region of CEP290 30 31 encompassing CCIII and part of the SMC homology domain (AAs 696 to 896) (Schafer et al., 32 33 2008). Combined knock-down of both proteins in zebrafish embryos synergistically augmented Deleted : 34 Knock-down assays in phenotypes seen in embryos treated with morpholinos for either NPHP5 or CEP290 . In Xenopus zebrafish embryos of either NPHP5 or 35 CEP290 leads to very similar 36 neurological abnormalities and laevis , expression of the NPHP5-binding domain of CEP290 caused substantial neural tube pronephric cysts, while combined knock- 37 down of both proteins synergistically 38 augmented these phenotypes . closure defects that were similar to the effect of NPHP5 knock-down. Moreover, co-expression 39 40 of the NPHP5-binding domain of CEP290 with NPHP5 itself rescued the phenotype, supporting a 41 42 physical interaction between both proteins in vivo (Schafer et al., 2008). 43 44 Deleted : MKS3 45 Both TMEM67 (MKS3) and CC2D2A are also CEP290-interacting proteins involved in disease. Deleted : add to 46 Deleted : that are 47 Mutations in both genes may cause MKS, JS and COACH syndrome, which are all severe Deleted : are 48 49 14 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 15 of 51 Human Mutation

1 2 ciliopathies with multi-systemic involvement (Smith et al., 2006; Baala et al., 2007b; Gorden et 3 Deleted : MKS3 4 al., 2008; Tallila et al., 2008; Brancati et al., 2009; Doherty et al., 2009). TMEM67 is involved in 5 6 both ciliary structure/function and endoplasmic reticulum-associated degradation of surfactant 7 8 protein C (Tammachote et al., 2009; Wang et al., 2009). Morpholino experiments in zebrafish 9 Deleted : mks3 10 revealed a strong genetic interaction between and Tmem67 , with more severe phenotypic 11 Deleted : proteins 12 effects following knock-down of both genes , compared with depletion of only one of both (Leitch 13 Deleted : is expressed in many adult 14 tissues, with high levels in prostate, et al., 2008). CC2D2A is presumed to act as a sensor for intracellular calcium and is part of the pancreas, lung, retina, kidney and fetal 15 brain and kidney (Gorden et al., 2008; 16 complex from which the cilium is assembled, where it co-localizes with CEP290 Noor et al., 2008). It 17 18 (Gorden et al., 2008; Tallila etFor al., 2008). ThePeer interaction between Review both proteins involves the 998 19 Deleted : n 20 N-terminal AAs of CC2D2A and a fragment of CEP290 containing CCs IV-VI (AAs 703-1130). Deleted : internal 21 Deleted : , 22 A functional interaction was observed in the pronephros of sentinel zebrafish (Gorden et al., 23 24 2008). 25 26 Deleted : overall 27 The involvement of CEP290 in the above-mentioned ciliary complexes strongly supports a role in 28 29 ciliogenesis and ciliary trafficking. The (tissue-specific) access of its binding partners is possibly 30 31 regulated by conformational changes of CEP290, since both the N-terminal (AAs 1-695) and C- 32 33 terminal (AAs 1966-2479) domains are involved in homo- and heterodimeric interactions 34 35 (Schafer et al., 2008). 36 37 Animal models 38 39 40 So far, two naturally occurring animal models are known with mutations in cep290 : a pedigree of 41 Deleted : animal 42 Abyssinian cats and the rd16 mouse. Both models display AR progressive retinal degeneration, 43 44 albeit with a different age-of-onset , possibly related to a differential effect of both distinct cep290 45 Deleted : Contrary to pleiotropic effects 46 mutations. Interestingly , no associated cerebellar or renal abnormalities are present in both of CEP290 in human 47 48 49 15 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 16 of 51

1 2 models (Chang et al., 2006; Menotti-Raymond et al., 2007). The cat and mouse orthologues show 3 4 92.1% and 87% AA to human CEP290, respectively (Menotti-Raymond et Deleted : Ensembl 5 6 al., 2007) ( www.ensembl.org ). 7 8 Deleted : causal gene 9 The genetic defect for the Abyssinian cat retinal degeneration (rdAc) consists of a nucleotide 10 11 substitution in intron 50 that creates a strong canonical splice donor site (IVS50+9T>G), resulting 12 Deleted : 13 in a prolongation of exon 50 with 4bp, and a frameshift with a PTC three AAs downstream. The Deleted : the inclusion of an additional 14 4 bp 15 translated protein is truncated by 159 AAs, corresponding to the loss of the KIDV and KIDVI Deleted : -insertion 16 Deleted : at the end of 17 domains (Menotti-Raymond et al., 2007). The age of onset of the first ophthalmoscopic signs is 18 For Peer Review 19 highly variable, with 12-18 months of age in the majority of animals. Full-field flash 20 Deleted : m 21 electroretinogra phy (ERG) at the age of eight months shows a reduction of mainly a-wave 22 23 amplitudes , whereas reduced sensitivity of the pupillary light reflex is observed with disease Deleted : The age of onset of the first 24 ophthalmoscopic signs is highly variable, 25 progression . (Thompson et al. , 2010 ; Narfstrom et al., 2009). Vacuolization and degeneration of with 12-18 months of age in the majority 26 of animals 27 membranes in the basal part of the rod outer segments ( OS ) in early stages of disease point to 28 29 potential defects in protein transport through the connecting cilia (Menotti-Raymond et al., 2007). 30 31 In contrast to rdAC, the rd16 mouse displays an early-onset retinal dystrophy, caused by a 32 Deleted : a 33 homozygous in-frame deletion of 897 bp (AAs 1599-1897) that covers the majority of the 34 35 myosin-tail homology region. Early fundus examinations revealed white retinal vessels and large 36 Deleted : functions 37 pigment patches, in addition to severely reduced ERG responses for both rod s and cone s. The 38 39 mutation causes early progressive degeneration of the OS and reduction in thickness of the outer 40 41 nuclear layer (Chang et al., 2006). In addition, considerable thickening of the inner nuclear and 42 43 plexiform layer were observed in mid- and central retinal regions, together with an enlargement 44 45 of nuclei of all retinal cell types (Cideciyan et al., 2007). Interestingly, rd16 mice displayed 46 Deleted : displayed 47 severe early-onset olfactory dysfunction. Although the olfactory sensory neurons have 48 49 16 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 17 of 51 Human Mutation

1 Deleted : ACIII, CNGA2, and different 2 structurally intact cilia with correct localization of CEP290 to dendritic knobs , two components G protein-coupled olfactory receptors 3

4 of the olfactory G protein that are in complex with CEP290 , Gγ13 and G olf , were undetectable in 5 Deleted : . These data sugges t 6 the cilia of rd16 mice , suggesting a potential role for CEP290 in the regulation of olfactory G Deleted : into cilia 7 8 protein trafficking (McEwen et al., 2007). 9 10 11 Both models offer interesting opportunities for in vivo research on CEP290 function and 12 13 therapeutic intervention. The progressive nature of retinal disease in rd16 mice resembles human 14 15 LCA, and therefore constitutes an excellent basis to study disease development. Moreover, the 16 17 existence of a large animal model such as rdAC is an asset for the implementation of gene- 18 For Peer Review 19 specific therapy for retinal degeneration, as already shown by successful RPE65 -replacement 20 Deleted : preceding 21 therapy in Briard dogs which preceded human phase I clinical trials (Acland et al., 2001; 22 23 Bainbridge et al., 2008; Hauswirth et al., 2008; Maguire et al., 2008; Maguire et al., 2009). 24 25 26 Clinical and diagnostic relevance 27 28 29 CEP290 -linked phenotypes 30 31 Leber Congenital Amaurosis (LCA) 32 33 34 Leber Congenital Amaurosis (LCA, MIM[204000]) is the earliest and most severe form of all 35 36 inherited retinal dystrophies, causing profound visual deficiency, nystagmus and an undetectable 37 38 or severely reduced ERG in the first year of life. Approximately 20% of all blind children are 39 Deleted : In general, LCA is inherited in 40 thought to suffer from this disease , which is mainly inherited in an AR manner . So far, 15 disease an AR manner. 41 Deleted : approximately 42 loci (14 genes) are known, which together account for ~70% of cases (den Hollander et al., 43 Deleted : causal 44 2008). In 2006, CEP290 was identified as a disease gene for LCA, with the deep intronic 45 Deleted : the initial 46 substitution c.2991+1655A>G as causal mutation. Targeted screening of this mutation in 76 47 48 49 17 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 18 of 51

1 Deleted : mainly originating from 2 unrelated LCA patients revealed its presence in 16 additional probands (21%), suggesting a major Northern Europe and Canada, 3 Deleted : 4 involvement of CEP290 in LCA (den Hollander et al., 2006). Subsequent studies corroborate that 5 Deleted : indeed 6 CEP290 mutations are highly recurrent in Northwestern Europe (Perrault et al., 2007; Coppieters 7 8 et al., 2010), but contribute to only a minor part of LCA in Italy, Saudi Arabia, Spain, Southern 9 Deleted : with a 10 India and Korea (targeted screening of c.2991+1655A>G in the latter three populations) 11 12 (Simonelli et al., 2007; Vallespin et al., 2007; Seong et al., 2008; Li et al., 2009; Sundaresan et 13 14 al., 2009). Interestingly, c.2991+1655A>G was not identified in 126 Spanish patients with early- 15 16 onset RP, suggesting specificity of this mutation for LCA (Vallespin et al., 2007). 17 18 For Peer Review 19 The CEP290 -related phenotype consists of a severe cone-rod type retinal dystrophy (Perrault et 20 21 al., 2007), with best-corrected visual acuity of counting fingers or worse in the majority of cases 22 23 (Walia et al. , 2010 ). Early fundus changes include white dots or a marbleized or salt and pepper 24 25 aspect, and progress to midperipheral nummular or spicular pigmentation (Littink et al. , 2010 ; 26 27 den Hollander et al., 2006; Perrault et al., 2007; Coppieters et al., 2010). A detailed study of the 28 29 retinal architecture of human CEP290 -mutant identified profound retinal remodeling in Deleted : 30 abnormal expansion 31 the peripheral rod-rich regions, which was characterized by thickening of inner retinal layers. In Deleted : 32 degeneration or thinning was 33 the cone-rich foveal region, however, no clear alterations were observed. This difference in rod Deleted : n 34 35 and cone degeneration may point to a distinct function of CEP290 in both cell types (Cideciyan et 36 37 al., 2007). Spectral-domain optical coherence tomography confirmed preservation of the outer Deleted : 38 r nuclear layer in the central macula with distorted inner retina (Pasadhika et al., 2009). No Deleted : area 39 40 Deleted : in six out of seven additional abnormalities were seen in visual brain pathway anatomy (Cideciyan et al., 2007). Apart from the patients 41 42 ocular phenotype, neurological involvement (mental retardation [MR] or ) was observed in 43 Deleted : 5 44 approximately 1 1-33% of cases with CEP290 -related LCA, which is more frequent than in 45 46 patients with mutations in other LCA genes (Hanein et al., 2004; Perrault et al., 2007; Coppieters 47 48 49 18 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 19 of 51 Human Mutation

1 2 et al., 2010). Four probands presented with either (transitory) hypotonia or ataxia (Perrault et al., 3 4 2007). Interestingly, some patients displayed signs suggestive for involvement of other ciliary 5 6 processes. One patient presented with recurrent otitis media (Coppieters et al., 2010), and LCA 7 8 patients homozygous for c.2991+1655A>G exhibited severe olfactory dysfunction, whereas mild 9 10 to severe microsmia was observed in heterozygous carriers (McEwen et al., 2007). Obviously, 11 12 MR, autism, ataxia and hypotonia may be signs of the mild end of the clinical spectrum of 13 14 systemic phenotypes related to CEP290 rather than of true LCA as isolated retinal disease. 15 16 17 Nephronophthisis (NPHP) and Senior-Loken syndrome (SLS) 18 For Peer Review 19 Nephronophthisis (NPHP, MIM[256100]) is the most frequent genetic cause for end-stage renal 20 21 disease (ESRD) in the first three decades of life. Infantile, juvenile and adolescent forms have 22 Deleted : depending on the age of onset 23 of ESRD been described. Key histology findings are tubulointerstitial fibrosis, tubular atrophy, tubular 24 25 dilatation and cyst formation. Renal ultrasound (US) reveals increased cortical echogenicity, loss 26 27 of corticomedullary differentiation and corticomedullary cysts (Salomon et al., 2009; Simms et 28 29 al., 2009). In 10-15% of cases, juvenile NPHP is accompanied by retinal involvement, which is 30 31 called Senior-Loken syndrome (SLS, MIM[266900]). Based on the degree of retinal 32 33 degeneration, both early-onset and late-onset types have been described. NPHP and SLS are AR 34 Deleted : six 35 disorders. So far, nine genes are known to cause NPHP; some of them are associated with SLS 36 37 (Hildebrandt and Zhou, 2007; Hildebrandt et al., 2009). CEP290 mutations have been described 38 39 in eight families with SLS, two of which present with intrafamilial clinical variability of 40 Deleted : , respectively 41 neurological and /or renal involvement (Tory et al., 2007). In six families, two mutations 42 43 segregated, while in two other families, one heterozygous CEP290 mutation occurred in 44 45 combination with a homozygous mutation in NPHP1 and a heterozygous mutation in NPHP4 , 46 Deleted : ESRD 47 respectively (Helou et al., 2007; Tory et al., 2007). The age at which ESRD occurred ranged from Deleted : between 48 49 19 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 20 of 51

1 Deleted : and 2 5 to 40 years and retinal degeneration was usually severe (Sayer et al., 2006; Helou et al., 2007; Deleted : 34 3 Deleted : One exception is patient F848, 4 Tory et al., 2007; Coppieters et al., 2010). It is presumed that CEP290 mutations do not represent heterozygous for one NPHP4 and one CEP290 mutation, who displayed ESRD 5 at the age of 40 and late-onset progressive 6 a major cause of NPHP/SLS, since large-scale mutation screening in almost 100 families with RP (Helou et al., 2007). 7 Deleted : , 8 SLS (Helou et al., 2007; O'Toole et al., 2007) and 21 families with isolated NPHP revealed 9 10 mutations in only two patients with SLS and a heterozygous unclassified variant in one patient 11 12 with NPHP (Helou et al., 2007). 13 14 15 Joubert syndrome (JS) and Joubert syndrome related disorders (JSRD) 16 Deleted : AR 17 Joubert syndrome (JS, MIM[213300]) is a genetically heterogeneous disorder of which the main 18 For Peer Review 19 phenotypic hallmarks are hypotonia, ataxia, psychomotor delay and variable occurrence of 20 21 oculomotor apraxia and neonatal breathing abnormalities. The most consistent feature , the “molar 22 Deleted : a unique constellation of 23 midbrain-hindbrain abnormalities, seen tooth sign” (MTS), is visible on magnetic resonance imaging (MRI) and consists of a deep 24 Deleted : . 25 interpeduncular fossa with narrow isthmus, thickened, elongated and mal-oriented superior Deleted : It is called “molar tooth sign” 26 (MTS) Deleted : 27 cerebellar peduncles and cerebellar vermis aplasia/hypoplasia (Doherty, 2009). In addition to essentially 28 29 pure JS, a range of disorders exists that share the MTS and the main neurological signs of JS, yet 30 Deleted : from JS 31 are distinct due to the involvement of other organs. They are called Joubert syndrome-related 32 Deleted : . 33 disorders (JSRD) and consist of five major subgroups: JS associated with retinopathy, JS Deleted : Based on additional features, 34 Valente and coworkers defined the following five subgroups: 35 associated with renal involvement, cerebello-oculo-renal syndrome (CORS or JS-SLS), COACH 36 37 syndrome (MIM[216360]) and orofaciodigital syndrome type VI (OFDVI, MIM[277170]) 38 39 (Valente et al., 2008). 40 41 Deleted : L 42 Currently, less than 50% of AR cases can be attributed to mutations in nine genes, which all Deleted : seven 43 Deleted : (nine l oci) 44 encode ciliary proteins , as far as known (Valente et al., 2008; Bielas et al., 2009; Doherty, 2009; 45 46 Valente et al., 2010). CEP290 mutations are a major cause of JSRD, in particular the CORS 47 48 49 20 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 21 of 51 Human Mutation

1 2 subgroup. Approximately 50% of cases with CORS are associated with CEP290 mutations. In 3 4 contrast, only a small fraction (~20%) of CEP290 mutations contributes to the other JSRD 5 Deleted : patients 6 subgroups (Valente et al., 2008). So far, only two families with classic JS harbored CEP290 7 Deleted : one 8 mutations, one of which displayed intrafamilial variability of the renal phenotype (Valente et al., 9 10 2006; Brancati et al., 2007). In most cases, NPHP was of the juvenile type, with development of 11 12 renal failure toward the end of the first decade or early in the second decade , whereas retinal 13 Deleted : However, renal failure at an 14 age of 4 and 25 was also reported degeneration mostly consisted of congenital blindness . Interestingly, several patients displayed (Brancati et al., 2007; Tory et al., 2007). 15 Retinal degeneration mostly consisted of 16 congenital blindness, although RP has associated features reminiscent of other subgroups or overlapping ciliopathies , including cleft also been described in a few patients 17 (Brancati et al., 2007; Helou et al., 2007). 18 palate, abnormal liver ultrasound,For elevated Peer liver enzymes, Review Hirschsprung disease, occipital Deleted : . These features include 19 20 (meningo)encephalocele, atrial/ventricular septal defects, cardiomegaly, postaxial polydactyly, 21 22 situs inversus , lobulated tongue, retinal coloboma, empty sella, hepatic fibrosis , recurrent otitis 23 24 media and hearing loss (Valente et al., 2006; Brancati et al., 2007; Helou et al., 2007; Tory et al., 25 26 2007; Coppieters et al., 2010). 27 28 29 Meckel-Grüber syndrome (MKS) 30 31 Meckel-Grüber syndrome (MKS, MIM[249000]) is a neonatally lethal disorder characterized by 32 33 a combination of central nervous system malformations (typically occipital 34 35 [meningo]encephalocele), bilateral cystic kidney dysplasia, ductal proliferation in the portal area 36 37 of the liver and postaxial polydactyly. Associated features may include cardiac abnormalities, 38 39 developmental anomalies of external or internal genitalia, cleft palate, situs inversus and others. 40 41 The worldwide incidence ranges from 1/13,250 to 1/140,000 live births (Alexiev et al., 2006). 42 43 Some cases present with incomplete phenotypes that are called “Meckel-like syndrome” 44 45 (MIM[208540]) and probably make up a clinical spectrum situated between JSRD and MKS. In 46 47 addition, a phenotypic and genetic overlap was seen between MKS and BBS (see “Bardet-Biedl 48 49 21 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 22 of 51

1 2 syndrome (BBS)”) (Karmous-Benailly et al., 2005; Leitch et al., 2008). MKS is inherited in an 3 4 AR fashion with five genes (six loci) identified so far, all encoding centrosomal/ciliary proteins. 5 6 Mutations in CEP290 were identified in eight families with MKS and four families with Meckel- 7 8 like syndrome, further supporting a role for CEP290 in a wide range of phenotypes (Baala et al., 9 10 2007a; Frank et al., 2008). 11 12 13 Bardet-Biedl syndrome (BBS) 14 15 Bardet-Biedl syndrome (BBS, MIM[209900] is a complex multi-organ , characterized 16 17 by a variable combination of retinal degeneration, obesity, hypogonadism, polydactyly, renal 18 For Peer Review 19 dysfunction and MR. Additional features consist of neurological impairment, speech deficits, 20 21 craniofacial abnormalities, hearing loss, diabetes mellitus, metabolic defects, cardiovascular 22 Deleted : in 23 abnormalities, hepatic defects and Hirschsprung disease. In most cases, BBS is inherited as an 24 Deleted : fashion 25 AR trait , with currently 14 genes known . Notably, mutations in different genes have been Deleted : for which 26 Deleted : loci 27 described in the same patient. These modifiers affect either the expressivity or the overall 28 Deleted : are known 29 penetrance of the phenotype (Zaghloul and Katsanis, 2009). In search of a genetic explanation for 30 31 the partial clinical overlap between BBS and MKS, Leitch and coworkers identified MKS1 and 32 33 CEP290 as disease genes for BBS. Interestingly, the homozygous p.Glu1903X CEP290 mutation 34 Deleted : MKS3 35 causing BBS was found together with a complex TMEM67 allele, possibly influencing the 36 37 phenotype (Leitch et al., 2008). 38 39 40 Genotype–phenotype correlations 41 42 43 For the majority of mutations, no clear-cut correlation could be established between the genotype 44 Deleted : 89 45 and clinical expression. Despite 90 mutations reported exclusively in only one phenotype, 14 46 47 others segregated with two diseases while 8 were even associated with three or more phenotypes. 48 49 22 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 23 of 51 Human Mutation

1 2 In most cases, these phenotypes are partially overlapping, although few mutations were observed 3 Deleted : in 4 to lead to strongly divergent disorders, such as LCA and MKS. Overall, mutations causing Deleted : M 5 6 JS(RD) tend to cluster in the second half of the gene, whereas mutations segregating with LCA, 7 8 SLS and MKS are homogeneously distributed throughout the gene (Brancati et al., 2007; Valente Deleted : Table 1 9 10 et al., 2008) ( Supp. Table S1 ). 11 12 13 For a few mutations, a genotype-phenotype correlation could be established, albeit to a limited 14 Deleted : Interestingly, t 15 extent. The most common mutation in JSRD, p.Gly1890X, was also described in two siblings 16 17 with isolated LCA, in compound heterozygosity with c.2991+1655A>G (Cideciyan et al., 2007). 18 For Peer Review 19 Despite the severe retinal phenotype of these siblings and four other patients heterozygous for 20 21 Gly1890X, a milder or even absent visual impairment was seen in eight of nine patients 22 23 homozygous for p.Gly1890X, suggesting that this mutation might be less harmful for retinal 24 25 function (Sayer et al., 2006; Valente et al., 2006; Brancati et al., 2007; Valente et al., 2008). 26 27 Similarly, a compound heterozygous genotype c.2991+1655G>A/c.451C>T caused an early- 28 29 onset retinal dystrophy phenotype less severe than most CEP290 -related LCA, probably due to Deleted : 30 the 31 the mild effect of both mutations (Littink et al. , 2010 ). So far, c.2991+1655A>G has only been Deleted : mutation 32 33 described in patients with isolated LCA. Since a small amount of wild-type product is still 34 35 present, a dosage-dependent mechanism was proposed, in which complete loss of function of 36 37 both alleles would lead to JS, whereas a residual CEP290 activity would be sufficient for normal 38 cerebellar and renal function but not for correct retinal activity (den Hollander et al., 2006). 39 40 However, subsequent identification of truncating mutations on both alleles in several LCA 41 42 patients countered the latter hypothesis (Perrault et al., 2007). In addition, we identified 43 Deleted : for 44 c.2991+1655A>G in two patients with features suggestive of renal dysfunction, implicating that 45 46 this mutation is possibly not exclusively associated with isolated LCA (Coppieters et al., 2010). 47 48 49 23 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 24 of 51

1 Deleted : stated 2 A similar hypothesis was proposed for certain other CEP290 splice site mutations. It was 3 4 presumed that these do not lead to truncated proteins, but rather to (partial) deletion of one of the 5 Deleted : Therefore 6 CCs. In contrast to truncating mutations , these do not seem to hamper normal neurologic Deleted : they 7 Deleted : might allow 8 development. Examples are two first-degree cousins, each carrying p.Leu1884ThrfsX23 on one Deleted : , in contrast to truncating 9 mutations 10 allele, but a different CEP290 mutation on the other. One of them harbored a nonsense mutation Deleted : This is based on clinical 11 findings in 12 and presented with JS without renal involvement by the age of eight, whereas the other was Deleted : one 13 14 heterozygous for c.4195-1G>A and suffered from SLS without neurologic symptoms (Tory et al., 15 16 2007). In addition, a homozygous splice site mutation segregated with SLS in a Turkish family 17 18 (Sayer et al., 2006). However,For in two other unrelatPeered patients Reviewwith neurological involvement, two 19 20 distinct splice site mutations were detected. Of note, in both cases, one of both mutations does not 21 22 affect a consensus splice site (c.1711+5A>G and c.6271-8T>G) (Perrault et al., 2007; Tory et al., 23 24 2007). 25 26 27 For all CEP290 -related phenotypes, different degrees of neurological, ocular and renal 28 29 involvement were observed between unrelated patients harboring the same CEP290 genotype. A 30 31 homozygous p.Lys1575X mutation was detected in four patients with isolated LCA and normal 32 33 development, one patient with LCA and autistic behavior but normal MRI, one patient with LCA Deleted : 34 for 35 and severe MR ( of whom no MRI was available), and one patient with LCA-JS (proven MTS on 36 37 MRI) (Perrault et al., 2007; Coppieters et al., 2010). Patients homozygous for p.Gly1890X Deleted : 38 for always displayed characteristics typical of JS but associated features ranged from none over JS Deleted : (isolated JS) 39 40 with NPHP to CORS (Sayer et al., 2006; Valente et al., 2006; Brancati et al., 2007). A 41 42 homozygous p.Trp7Cys mutation was identified in two patients of Pakistani origin who suffered 43 44 from retinal degeneration and NPHP. In only one patient, however, several features suggestive 45 46 for JS were observed (Valente et al., 2006; Coppieters et al., 2010). In addition, compound 47 48 49 24 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 25 of 51 Human Mutation

1 2 heterozygous p.Leu1884ThrfsX23 and p.Phe1950LeufsX15 mutations were described in two 3 4 French siblings with CORS (Tory et al., 2007), and two French siblings with Meckel-like 5 6 syndrome, aged 18 and 29 weeks of gestation, respectively (Baala et al., 2007a). Moreover, 7 8 intrafamilial variability was reported in several cases. Perrault and colleagues described two LCA 9 10 families in which all affected sibs carried the same mutations, but displayed different 11 Deleted : . 12 neurological involvement (Perrault et al., 2007). In addition, kidney US in two brothers with JS Deleted : In the first family, only one of 13 two affected sibs suffered from autism, whereas in the other family, two out of 14 (deceased at an age of four and seven months), both homozygous for p.Gln1942X, revealed five affected sibs were mentally retarded 15 16 cortical cysts in only one patient (Valente et al., 2006). Overall, this wide clinical spectrum is 17 18 difficult to explain by the CEP290For genotype Peer alone. Review 19 20 21 Epistatic effect of other components of the ciliary proteome 22 23 It is presumed that the clinical variability of CEP290 -related disease might be caused by second- 24 Deleted : is in 25 site modifier alleles. Since CEP290 forms a complex with several members of the ciliary 26 27 proteome, variants in these genes are likely to affect the interaction with and function of CEP290. 28 29 So far, three ciliary genes have been described in which variants co-occur with CEP290 30 31 mutations. The first gene is AHI1 , which is associated with JS(RD) and possibly acts in a 32 Deleted : pathway as 33 pathway common with CEP290 (Ferland et al., 2004; Parisi et al., 2006; Kim et al., 2008; Hsiao 34 35 et al., 2009). Tory and colleagues identified a heterozygous p.Arg830Trp missense variant in a 36 37 CORS patient harboring a homozygous p.Leu1884ThrfsX23 CEP290 mutation. This AHI1 38 39 variant was not present in an affected sibling with the same CEP290 genotype. Interestingly, no 40 41 significant difference could be seen in neurological and ocular manifestation between both sibs, 42 43 albeit that the one carrying p.Arg830Trp displayed renal failure earlier than the other (11 versus 44 45 25 years). In addition, a higher frequency of p.Arg830Trp was observed in patients with NPHP1 46 47 mutations and neurological symptoms, in comparison with patients with NPHP1 mutations 48 49 25 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 26 of 51

1 2 lacking neurological involvement or with healthy controls, suggesting an influence on the 3 Deleted : and 4 neurological expression of NPHP1 -related disease. Mutations in NPHP1 cause NPHP or SLS , 5 Deleted : Very 6 with JS-related neurological involvement in up to 12% of cases (Tory et al., 2007). Recently, a Deleted : r 7 8 modifying effect of p.Arg830Trp has also been established on the development of retinal 9 10 degeneration in patients suffering from NPHP, independent of primary mutations in NPHP1 . 11 Deleted : Patients with NPHP carrying 12 p.Arg830Trp have a relative risk of 7.5 AHI1 genetically interacts with NPHP1 in retinal development and was proven necessary for (95% CI 4.0–11.2) to develop retinal 13 degeneration. 14 photoreceptor OS development (Louie et al. , 2010 ). In addition to p.Arg830Trp, we identified a 15 16 different heterozygous missense variant, p.Asn811Lys, in the most severely affected patient out 17 18 of three with the same CEP290For genotype andPeer LCA but with Reviewdifferent neurological involvement 19 Deleted : A 20 as well as variable age-of-onset of ESRD. Yet another LCA patient with MR carried a third AHI1 21 22 missense variant, p.His758Pro, in combination with two mutations in CEP290 (Coppieters et al., 23 Deleted : are 24 2010). Of note, all potential modifier alleles identified in AHI1 so far have been missense 25 26 variants , located in the strongly conserved WD40 repeat, whereas most of the JS-causing 27 28 mutations are truncating. Obviously, these variants in AHI1 are not sufficient to explain all of the 29 Deleted : could be 30 clinical variability, as no AHI1 mutations were demonstrated in two large cohorts of patients with 31 Deleted : , 32 JS(RD) and CEP290 mutations (Brancati et al., 2007; Helou et al., 2007). A second gene Deleted : suggesting the involvement of 33 other factors 34 considered to harbor a potential modifier allele is TMEM67 ( MKS3 ). A homozygous p.Glu1903X Deleted : The 35 Deleted : MKS3 36 CEP290 mutation was accompanied by a complex p.[Gly218Ala; Ser320Cys] TMEM67 allele in Deleted : TMEM67 37 Deleted : MKS3 38 a patient with BBS. The TMEM67 allele is likely to influence the CEP290 -related phenotype, Deleted : MKS3 39 Deleted : mks 3 40 given the genetic interaction between cep290 and Tmem67 in zebrafish and the pathogenic 41 42 potential of the p.Ser320Cys variant and to a lesser extent the p.Gly218Ala variant (Leitch et al., 43 Deleted : makes up 44 2008). NPHP4 , which is associated with both isolated NPHP and SLS (Otto et al., 2002), is the 45 46 third gene, since a heterozygous p.Thr627Met mutation in NPHP4 was identified in a patient 47 48 with SLS who is also heterozygous for p.Arg1978X in CEP290 (Hoefele et al., 2005; Helou et 49 26 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 27 of 51 Human Mutation

1 Deleted : MKS3 2 al., 2007) . In addition to the heterozygous alleles identified in AHI1 , TMEM67 and NPHP4 , both 3 4 homozygosity at the PKHD1 locus and a homozygous CEP290 mutation were identified in a 5 6 consanguineous family segregating MKS as well as AR polycystic kidney disease. As expected, a 7 8 homozygous CEP290 mutation combined with linkage to the PKHD1 locus in one sibling with 9 10 MKS caused a more severe kidney and liver phenotype, in comparison with the other siblings 11 12 that carried either the same CEP290 or PKHD1 genotype (Baala et al., 2007a). 13 14 15 Conversely, mutations in CEP290 might affect phenotypes caused by mutations in other ciliary 16 Deleted : that 17 genes. A heterozygous p.Asn96MetfsX29 mutation segregated in two siblings who also harbored 18 For Peer Review 19 a homozygous NPHP1 deletion. In spite of a common genotype and similar retinal degeneration, 20 21 these siblings differ in both renal and neurological phenotype, suggesting the involvement of 22 23 additional factors (Tory et al., 2007). In addition, a heterozygous CEP290 mutation/variant was 24 25 identified in two probands that might carry a homozygous mutation in another ciliary gene, since 26 27 they each originate from consanguineous parents (Helou et al., 2007). 28 29 Molecular diagnostic strategies 30 31 32 Given the large number of coding exons (53), sequencing of CEP290 is laborious, expensive and 33 34 requires a considerable amount of DNA. Therefore, first-step analysis might comprise screening 35 36 for the most recurrent mutations, the choice of which depends on the phenotype of the patient. 37 38 This approach has been applied successfully by several groups (den Hollander et al., 2006; 39 Deleted : done 40 Perrault et al., 2007; Coppieters et al., 2010). Targeted mutation screening can be performed 41 Deleted : by 42 using denaturing high-performance liquid chromatography (dHPLC) or sequencing. For a few Deleted : , 43 Deleted : , or by other means 44 mutations, specific detection techniques are available, such as an allele-specific PCR for 45 46 c.2991+1655A>G (den Hollander et al., 2006). Moreover, 96 CEP290 mutations are present on a 47 48 49 27 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 28 of 51

1 Deleted : LCA 2 commercially available microarray, which represents standard genetic screening for LCA by 3 Deleted : early-onset RP and LCA 4 testing for 641 mutations in 13 genes (Asper Ophthalmics, v8.0). In the majority of cases 5 6 however, screening of the complete coding region remains necessary, either to identify the 7 8 second disease allele, or to screen for both mutations in case of negative first-pass screening. 9 10 PCR primers and conditions for both sequencing (Sayer et al., 2006; Valente et al., 2006) and 11 Deleted : purposes 12 dHPLC (Brancati et al., 2007) have been published and an alternative pre-screening assay based 13 14 on heteroduplex formation and subsequent CEL I endonuclease digest has been described (Helou 15 Deleted : In order to preserve DNA of 16 et al., 2007). Brancati and colleagues first screened parental DNA using dHPLC, followed by affected children, 17 18 sequencing of the identified mutationsFor in thePeer affected offspring Review and of the whole coding region in 19 20 case of a heterozygous mutation (Brancati et al., 2007). Apart from detecting small variants, 21 22 comprehensive CEP290 screening should also include dosage analysis of all exons, using for 23 24 instance quantitative real-time PCR (Travaglini et al., 2009). 25 26 27 Future Prospects 28 29 30 Given the emerging importance of modifier alleles on the phenotypic expression of ciliopathies, 31 Deleted : necessary 32 future studies are required to understand their mechanism of action and to further elucidate the 33 34 ciliary protein networks. The identification of modifiers for retinal, renal, neurological or other 35 36 phenotypes might contribute to an improved predictive power of a CEP290 -related genotype. 37 38 Early management of patients with a higher risk of NPHP, for instance, may delay the 39 Deleted : clinical 40 progression toward renal failure and minimize secondary complications. In a diagnostic setting, 41 42 mutations in ciliopathies should ideally be evaluated in the context of the entire spectrum of 43 44 ciliary variants. Novel technologies such as next-generation sequencing will play a crucial role in 45 46 this respect. Finally, more insights in the molecular pathogenesis of ciliopathies will eventually 47 48 direct towards therapeutic options, such as gene therapy. Since all CEP290 -related phenotypes 49 28 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 29 of 51 Human Mutation

1 Deleted : autosomal recessive 2 are inherited in an AR manner and the majority of mutations consist of loss-of-function alleles, 3 4 gene-replacement therapy might indeed be an option. 5 6 7 References 8 9 10 Acland GM, Aguirre GD, Ray J, Zhang Q, Aleman TS, Cideciyan AV, Pearce-Kelling SE, 11 Anand V, Zeng Y, Maguire AM, Jacobson SG, Hauswirth WW, Bennett J. 2001. Gene 12 therapy restores vision in a canine model of childhood blindness. Nat Genet 28(1):92-5. 13 Alexiev BA, Lin X, Sun CC, Brenner DS. 2006. Meckel-Gruber syndrome: pathologic 14 manifestations, minimal diagnostic criteria, and differential diagnosis. Arch Pathol Lab 15 Med 130(8):1236-8. 16 Ameri K, Harris AL. 2008. Activating transcription factor 4. Int J Biochem Cell Biol 40(1):14- 17 21. 18 Andersen JS, Wilkinson CJ, MayorFor T, Mortensen Peer P, Nigg EA, Review Mann M. 2003. Proteomic 19 characterization of the human centrosome by protein correlation profiling. Nature 20 426(6966):570-4. 21 Baala L, Audollent S, Martinovic J, Ozilou C, Babron MC, Sivanandamoorthy S, Saunier S, 22 Salomon R, Gonzales M, Rattenberry E, Esculpavit C, Toutain A, Moraine C, Parent P, 23 Marcorelles P, Dauge MC, Roume J, Le Merrer M, Meiner V, Meir K, Menez F, 24 Beaufrere AM, Francannet C, Tantau J, Sinico M, Dumez Y, MacDonald F, Munnich A, 25 Lyonnet S, Gubler MC, Genin E, Johnson CA, Vekemans M, Encha-Razavi F, Attie- 26 Bitach T. 2007a. Pleiotropic effects of CEP290 (NPHP6) mutations extend to Meckel 27 syndrome. Am J Hum Genet 81(1):170-9. 28 Baala L, Romano S, Khaddour R, Saunier S, Smith UM, Audollent S, Ozilou C, Faivre L, 29 Laurent N, Foliguet B, Munnich A, Lyonnet S, Salomon R, Encha-Razavi F, Gubler MC, 30 Boddaert N, de Lonlay P, Johnson CA, Vekemans M, Antignac C, Attie-Bitach T. 2007b. 31 The Meckel-Gruber syndrome gene, MKS3, is mutated in Joubert syndrome. Am J Hum 32 Genet 80(1):186-94. 33 Bainbridge JW, Smith AJ, Barker SS, Robbie S, Henderson R, Balaggan K, Viswanathan A, 34 Holder GE, Stockman A, Tyler N, Petersen-Jones S, Bhattacharya SS, Thrasher AJ, 35 Fitzke FW, Carter BJ, Rubin GS, Moore AT, Ali RR. 2008. Effect of gene therapy on 36 visual function in Leber's congenital amaurosis. N Engl J Med 358(21):2231-9. 37 Berbari NF, O'Connor AK, Haycraft CJ, Yoder BK. 2009. The primary cilium as a complex 38 signaling center. Curr Biol 19(13):R526-35. 39 Bielas SL, Silhavy JL, Brancati F, Kisseleva MV, Al-Gazali L, Sztriha L, Bayoumi RA, Zaki 40 MS, Abdel-Aleem A, Rosti RO, Kayserili H, Swistun D, Scott LC, Bertini E, Boltshauser 41 E, Fazzi E, Travaglini L, Field SJ, Gayral S, Jacoby M, Schurmans S, Dallapiccola B, 42 Majerus PW, Valente EM, Gleeson JG. 2009. Mutations in INPP5E, encoding inositol 43 polyphosphate-5-phosphatase E, link phosphatidyl inositol signaling to the ciliopathies. 44 Nat Genet 41(9):1032-6. 45 Brancati F, Barrano G, Silhavy JL, Marsh SE, Travaglini L, Bielas SL, Amorini M, Zablocka D, 46 Kayserili H, Al-Gazali L, Bertini E, Boltshauser E, D'Hooghe M, Fazzi E, Fenerci EY, 47 Hennekam RC, Kiss A, Lees MM, Marco E, Phadke SR, Rigoli L, Romano S, Salpietro 48 CD, Sherr EH, Signorini S, Stromme P, Stuart B, Sztriha L, Viskochil DH, Yuksel A, 49 29 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 30 of 51

1 2 Dallapiccola B, Valente EM, Gleeson JG. 2007. CEP290 mutations are frequently 3 identified in the oculo-renal form of Joubert syndrome-related disorders. Am J Hum 4 Genet 81(1):104-13. 5 Brancati F, Iannicelli M, Travaglini L, Mazzotta A, Bertini E, Boltshauser E, D'Arrigo S, Emma 6 F, Fazzi E, Gallizzi R, Gentile M, Loncarevic D, Mejaski-Bosnjak V, Pantaleoni C, Rigoli 7 L, Salpietro CD, Signorini S, Stringini GR, Verloes A, Zabloka D, Dallapiccola B, 8 Gleeson JG, Valente EM. 2009. MKS3/TMEM67 mutations are a major cause of COACH 9 Syndrome, a Joubert Syndrome related disorder with liver involvement. Hum Mutat 10 30(2):E432-42. 11 Chang B, Khanna H, Hawes N, Jimeno D, He S, Lillo C, Parapuram SK, Cheng H, Scott A, Hurd 12 RE, Sayer JA, Otto EA, Attanasio M, O'Toole JF, Jin G, Shou C, Hildebrandt F, Williams 13 DS, Heckenlively JR, Swaroop A. 2006. In-frame deletion in a novel centrosomal/ciliary 14 protein CEP290/NPHP6 perturbs its interaction with RPGR and results in early-onset 15 retinal degeneration in the rd16 mouse. Hum Mol Genet 15(11):1847-57. 16 Chen D, Shou C. 2001. Molecular cloning of a tumor-associated antigen recognized by 17 monoclonal antibody 3H11. Biochem Biophys Res Commun 280(1):99-103. 18 Cideciyan AV, Aleman TS, JacobsonFor SG, KhannaPeer H, Sumaroka Review A, Aguirre GK, Schwartz SB, 19 Windsor EA, He S, Chang B, Stone EM, Swaroop A. 2007. Centrosomal-ciliary gene 20 CEP290/NPHP6 mutations result in blindness with unexpected sparing of photoreceptors 21 and visual brain: implications for therapy of Leber congenital amaurosis. Hum Mutat 22 28(11):1074-83. 23 Coppieters F, Casteels I, Meire FM, De Jaegere S, Hooghe S, Van Regemorter N, Van Esch H, 24 Matulevi čien ÷ A, Castedo S, Meersschaut V, Walraedt S, Standaert L, Coucke P, Hoeben 25 H, Kroes HY, Vande Walle J, de Ravel T, Leroy BP, De Baere E. 2010. Genetic 26 screening of LCA in Belgium: predominance of CEP290 and identification of potential 27 modifier alleles in AHI1 of CEP290-related phenotypes. Under review . Deleted : Submitted 28 den Hollander AI, Koenekoop RK, Yzer S, Lopez I, Arends ML, Voesenek KE, Zonneveld MN, 29 Strom TM, Meitinger T, Brunner HG, Hoyng CB, van den Born LI, Rohrschneider K, 30 Cremers FP. 2006. Mutations in the CEP290 (NPHP6) gene are a frequent cause of Leber 31 congenital amaurosis. Am J Hum Genet 79(3):556-61. 32 den Hollander AI, Roepman R, Koenekoop RK, Cremers FP. 2008. Leber congenital amaurosis: 33 genes, proteins and disease mechanisms. Prog Retin Eye Res 27(4):391-419. 34 Doherty D. 2009. Joubert syndrome: insights into brain development, cilium biology, and 35 complex disease. Semin Pediatr Neurol 16(3):143-54. 36 Doherty D, Parisi MA, Finn LS, Gunay-Aygun M, Al-Mateen M, Bates D, Clericuzio C, Demir 37 H, Dorschner M, van Essen AJ, Gahl WA, Gentile M, Gorden NT, Hikida A, Knutzen D, 38 Ozyurek H, Phelps I, Rosenthal P, Verloes A, Weigand H, Chance PF, Dobyns WB, Glass 39 IA. 2009. Mutations in 3 genes (MKS3, CC2D2A and RPGRIP1L) cause COACH 40 syndrome (Joubert syndrome with congenital hepatic fibrosis). J Med Genet 47(1):8-21. Deleted : . 41 Dryja TP, Adams SM, Grimsby JL, McGee TL, Hong DH, Li T, Andreasson S, Berson EL. 2001. 42 Null RPGRIP1 alleles in patients with Leber congenital amaurosis. Am J Hum Genet 43 68(5):1295-8. 44 Ferland RJ, Eyaid W, Collura RV, Tully LD, Hill RS, Al-Nouri D, Al-Rumayyan A, Topcu M, 45 Gascon G, Bodell A, Shugart YY, Ruvolo M, Walsh CA. 2004. Abnormal cerebellar 46 development and axonal decussation due to mutations in AHI1 in Joubert syndrome. Nat 47 Genet 36(9):1008-13. 48 49 30 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 31 of 51 Human Mutation

1 2 Frank V, den Hollander AI, Bruchle NO, Zonneveld MN, Nurnberg G, Becker C, Du Bois G, 3 Kendziorra H, Roosing S, Senderek J, Nurnberg P, Cremers FP, Zerres K, Bergmann C. 4 2008. Mutations of the CEP290 gene encoding a centrosomal protein cause Meckel- 5 Gruber syndrome. Hum Mutat 29(1):45-52. 6 Gerdes JM, Davis EE, Katsanis N. 2009. The vertebrate primary cilium in development, 7 homeostasis, and disease. Cell 137(1):32-45. 8 Gorden NT, Arts HH, Parisi MA, Coene KL, Letteboer SJ, van Beersum SE, Mans DA, Hikida 9 A, Eckert M, Knutzen D, Alswaid AF, Ozyurek H, Dibooglu S, Otto EA, Liu Y, Davis 10 EE, Hutter CM, Bammler TK, Farin FM, Dorschner M, Topcu M, Zackai EH, Rosenthal 11 P, Owens KN, Katsanis N, Vincent JB, Hildebrandt F, Rubel EW, Raible DW, Knoers 12 NV, Chance PF, Roepman R, Moens CB, Glass IA, Doherty D. 2008. CC2D2A is 13 mutated in Joubert syndrome and interacts with the ciliopathy-associated basal body 14 protein CEP290. Am J Hum Genet 83(5):559-71. 15 Grantham R. 1974. Amino acid difference formula to help explain protein evolution. Science 16 185(4154):862-4. 17 Guo J, Jin G, Meng L, Ma H, Nie D, Wu J, Yuan L, Shou C. 2004. Subcellullar localization of 18 tumor-associated antigenFor 3H11Ag. BiochemPeer Biophys ReReviews Commun 324(2):922-30. 19 Hanein S, Perrault I, Gerber S, Tanguy G, Barbet F, Ducroq D, Calvas P, Dollfus H, Hamel C, 20 Lopponen T, Munier F, Santos L, Shalev S, Zafeiriou D, Dufier JL, Munnich A, Rozet 21 JM, Kaplan J. 2004. Leber congenital amaurosis: comprehensive survey of the genetic 22 heterogeneity, refinement of the clinical definition, and genotype-phenotype correlations 23 as a strategy for molecular diagnosis. Hum Mutat 23(4):306-17. 24 Hauswirth WW, Aleman TS, Kaushal S, Cideciyan AV, Schwartz SB, Wang L, Conlon TJ, Boye 25 SL, Flotte TR, Byrne BJ, Jacobson SG. 2008. Treatment of leber congenital amaurosis 26 due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene 27 vector: short-term results of a phase I trial. Hum Gene Ther 19(10):979-90. 28 Helou J, Otto EA, Attanasio M, Allen SJ, Parisi MA, Glass I, Utsch B, Hashmi S, Fazzi E, 29 Omran H, O'Toole JF, Sayer JA, Hildebrandt F. 2007. Mutation analysis of 30 NPHP6/CEP290 in patients with Joubert syndrome and Senior-Loken syndrome. J Med 31 Genet 44(10):657-63. 32 Hildebrandt F, Attanasio M, Otto E. 2009. Nephronophthisis: disease mechanisms of a ciliopathy. 33 J Am Soc Nephrol 20(1):23-35. 34 Hildebrandt F, Zhou W. 2007. Nephronophthisis-associated ciliopathies. J Am Soc Nephrol 35 18(6):1855-71. 36 Hoefele J, Sudbrak R, Reinhardt R, Lehrack S, Hennig S, Imm A, Muerb U, Utsch B, Attanasio 37 M, O'Toole JF, Otto E, Hildebrandt F. 2005. Mutational analysis of the NPHP4 gene in 38 250 patients with nephronophthisis. Hum Mutat 25(4):411. 39 Hong DH, Pawlyk B, Sokolov M, Strissel KJ, Yang J, Tulloch B, Wright AF, Arshavsky VY, Li 40 T. 2003. RPGR isoforms in photoreceptor connecting cilia and the transitional zone of 41 motile cilia. Invest Ophthalmol Vis Sci 44(6):2413-21. 42 Hsiao YC, Tong ZJ, Westfall JE, Ault JG, Page-McCaw PS, Ferland RJ. 2009. Ahi1, whose 43 human ortholog is mutated in Joubert syndrome, is required for Rab8a localization, 44 ciliogenesis and vesicle trafficking. Hum Mol Genet 18(20):3926-41. 45 Karmous-Benailly H, Martinovic J, Gubler MC, Sirot Y, Clech L, Ozilou C, Auge J, Brahimi N, 46 Etchevers H, Detrait E, Esculpavit C, Audollent S, Goudefroye G, Gonzales M, Tantau J, 47 Loget P, Joubert M, Gaillard D, Jeanne-Pasquier C, Delezoide AL, Peter MO, Plessis G, 48 Simon-Bouy B, Dollfus H, Le Merrer M, Munnich A, Encha-Razavi F, Vekemans M, 49 31 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 32 of 51

1 2 Attie-Bitach T. 2005. Antenatal presentation of Bardet-Biedl syndrome may mimic 3 Meckel syndrome. Am J Hum Genet 76(3):493-504. 4 Kim J, Krishnaswami SR, Gleeson JG. 2008. CEP290 interacts with the centriolar satellite 5 component PCM-1 and is required for Rab8 localization to the primary cilium. Hum Mol 6 Genet 17(23):3796-805. 7 Leitch CC, Zaghloul NA, Davis EE, Stoetzel C, Diaz-Font A, Rix S, Al-Fadhel M, Lewis RA, 8 Eyaid W, Banin E, Dollfus H, Beales PL, Badano JL, Katsanis N. 2008. Hypomorphic 9 mutations in syndromic encephalocele genes are associated with Bardet-Biedl syndrome. 10 Nat Genet 40(4):443-8. 11 Li Y, Wang H, Peng J, Gibbs RA, Lewis RA, Lupski JR, Mardon G, Chen R. 2009. Mutation 12 survey of known LCA genes and loci in the Saudi Arabian population. Invest Ophthalmol 13 Vis Sci 50(3):1336-43. 14 Littink KW, Pott JW, Collin RW, Kroes HY, Verheij JB, Blokland EA, de Castro Miro M, 15 Hoyng CB, Klaver C, Koenekoop RK, Rohrschneider K, Cremers FP, van den Born I, den 16 Hollander AI. 2010. A novel nonsense mutation in CEP290 induces exon skipping and 17 leads to a relatively mild retinal phenotype. Invest Ophthalmol Vis Sci. In Press. 18 Louie CM, Caridi G, Lopes VS,For Brancati F, PeerKispert A, Lancaster Review MA, Schlossman AM, Otto EA, 19 Leitges M, Grone HJ, Lopez I, Gudiseva HV, O'Toole JF, Vallespin E, Ayyagari R, 20 Ayuso C, Cremers FP, den Hollander AI, Koenekoop RK, Dallapiccola B, Ghiggeri GM, 21 Hildebrandt F, Valente EM, Williams DS, Gleeson JG. 2010. AHI1 is required for 22 photoreceptor outer segment development and is a modifier for retinal degeneration in 23 nephronophthisis. Nat Genet 42(2):175-80. Deleted : . 24 Maguire AM, High KA, Auricchio A, Wright JF, Pierce EA, Testa F, Mingozzi F, Bennicelli JL, 25 Ying GS, Rossi S, Fulton A, Marshall KA, Banfi S, Chung DC, Morgan JI, Hauck B, 26 Zelenaia O, Zhu X, Raffini L, Coppieters F, De Baere E, Shindler KS, Volpe NJ, Surace 27 EM, Acerra C, Lyubarsky A, Redmond TM, Stone E, Sun J, McDonnell JW, Leroy BP, 28 Simonelli F, Gauderman JB. 2009. Age-dependent effects of RPE65 gene therapy for 29 Leber's congenital amaurosis: a phase 1 dose-escalation trial. Lancet 375(9708):30. Deleted : . 30 Maguire AM, Simonelli F, Pierce EA, Pugh EN, Jr., Mingozzi F, Bennicelli J, Banfi S, Marshall 31 KA, Testa F, Surace EM, Rossi S, Lyubarsky A, Arruda VR, Konkle B, Stone E, Sun J, 32 Jacobs J, Dell'Osso L, Hertle R, Ma JX, Redmond TM, Zhu X, Hauck B, Zelenaia O, 33 Shindler KS, Maguire MG, Wright JF, Volpe NJ, McDonnell JW, Auricchio A, High KA, 34 Bennett J. 2008. Safety and efficacy of gene transfer for Leber's congenital amaurosis. N 35 Engl J Med 358(21):2240-8. 36 McEwen DP, Koenekoop RK, Khanna H, Jenkins PM, Lopez I, Swaroop A, Martens JR. 2007. 37 Hypomorphic CEP290/NPHP6 mutations result in anosmia caused by the selective loss of 38 G proteins in cilia of olfactory sensory neurons. Proc Natl Acad Sci U S A 39 104(40):15917-22. 40 Meindl A, Dry K, Herrmann K, Manson F, Ciccodicola A, Edgar A, Carvalho MR, Achatz H, 41 Hellebrand H, Lennon A, Migliaccio C, Porter K, Zrenner E, Bird A, Jay M, Lorenz B, 42 Wittwer B, D'Urso M, Meitinger T, Wright A. 1996. A gene (RPGR) with homology to 43 the RCC1 guanine nucleotide exchange factor is mutated in X-linked retinitis pigmentosa 44 (RP3). Nat Genet 13(1):35-42. 45 Menotti-Raymond M, David VA, Schaffer AA, Stephens R, Wells D, Kumar-Singh R, O'Brien 46 SJ, Narfstrom K. 2007. Mutation in CEP290 discovered for cat model of human retinal 47 degeneration. J Hered 98(3):211-20. 48 49 32 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 33 of 51 Human Mutation

1 2 Nagase T, Ishikawa K, Nakajima D, Ohira M, Seki N, Miyajima N, Tanaka A, Kotani H, Nomura 3 N, Ohara O. 1997. Prediction of the coding sequences of unidentified human genes. VII. 4 The complete sequences of 100 new cDNA clones from brain which can code for large 5 proteins in vitro. DNA Res 4(2):141-50. 6 Nagy E, Maquat LE. 1998. A rule for termination-codon position within intron-containing genes: 7 when nonsense affects RNA abundance. Trends Biochem Sci 23(6):198-9. 8 Narfstrom K, David V, Jarret O, Beatty J, Barrs V, Wilkie D, O'Brien S, Menotti-Raymond M. 9 2009. Retinal degeneration in the Abyssinian and Somali cat (rdAc): correlation between 10 genotype and phenotype and rdAc allele frequency in two continents. Vet Ophthalmol 11 12(5):285-91. 12 Nigg EA, Raff JW. 2009. Centrioles, centrosomes, and cilia in health and disease. Cell 13 139(4):663-78. 14 O'Toole JF, Otto EA, Hoefele J, Helou J, Hildebrandt F. 2007. Mutational analysis in 119 15 families with nephronophthisis. Pediatr Nephrol 22(3):366-70. 16 Otto E, Hoefele J, Ruf R, Mueller AM, Hiller KS, Wolf MT, Schuermann MJ, Becker A, 17 Birkenhager R, Sudbrak R, Hennies HC, Nurnberg P, Hildebrandt F. 2002. A gene 18 mutated in nephronophthisisFor and retinitis Peer pigmentosa encodesReview a novel protein, 19 nephroretinin, conserved in evolution. Am J Hum Genet 71(5):1161-7. 20 Otto EA, Loeys B, Khanna H, Hellemans J, Sudbrak R, Fan S, Muerb U, O'Toole JF, Helou J, 21 Attanasio M, Utsch B, Sayer JA, Lillo C, Jimeno D, Coucke P, De Paepe A, Reinhardt R, 22 Klages S, Tsuda M, Kawakami I, Kusakabe T, Omran H, Imm A, Tippens M, Raymond 23 PA, Hill J, Beales P, He S, Kispert A, Margolis B, Williams DS, Swaroop A, Hildebrandt 24 F. 2005. Nephrocystin-5, a ciliary IQ domain protein, is mutated in Senior-Loken 25 syndrome and interacts with RPGR and calmodulin. Nat Genet 37(3):282-8. 26 Parisi MA, Doherty D, Eckert ML, Shaw DW, Ozyurek H, Aysun S, Giray O, Al Swaid A, Al 27 Shahwan S, Dohayan N, Bakhsh E, Indridason OS, Dobyns WB, Bennett CL, Chance PF, 28 Glass IA. 2006. AHI1 mutations cause both retinal dystrophy and renal cystic disease in 29 Joubert syndrome. J Med Genet 43(4):334-9. 30 Pasadhika S, Fishman GA, Stone EM, Lindeman M, Zelkha R, Lopez I, Koenekoop RK, Shahidi 31 M. 2009. Differential Macular Morphology in Patients with RPE65, CEP290, GUCY2D 32 and AIPL1 Related Leber Congenital Amaurosis. Invest Ophthalmol Vis Sci 51(5):2608- 33 14. Deleted : . 34 Perrault I, Delphin N, Hanein S, Gerber S, Dufier JL, Roche O, Defoort-Dhellemmes S, Dollfus 35 H, Fazzi E, Munnich A, Kaplan J, Rozet JM. 2007. Spectrum of NPHP6/CEP290 36 mutations in Leber congenital amaurosis and delineation of the associated phenotype. 37 Hum Mutat 28(4):416. 38 Salomon R, Saunier S, Niaudet P. 2009. Nephronophthisis. Pediatr Nephrol 24(12):2333-44. 39 Sayer JA, Otto EA, O'Toole JF, Nurnberg G, Kennedy MA, Becker C, Hennies HC, Helou J, 40 Attanasio M, Fausett BV, Utsch B, Khanna H, Liu Y, Drummond I, Kawakami I, 41 Kusakabe T, Tsuda M, Ma L, Lee H, Larson RG, Allen SJ, Wilkinson CJ, Nigg EA, Shou 42 C, Lillo C, Williams DS, Hoppe B, Kemper MJ, Neuhaus T, Parisi MA, Glass IA, Petry 43 M, Kispert A, Gloy J, Ganner A, Walz G, Zhu X, Goldman D, Nurnberg P, Swaroop A, 44 Leroux MR, Hildebrandt F. 2006. The centrosomal protein nephrocystin-6 is mutated in 45 Joubert syndrome and activates transcription factor ATF4. Nat Genet 38(6):674-81. 46 Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner 47 M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A, 48 49 33 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 34 of 51

1 2 Walz G. 2008. Genetic and physical interaction between the NPHP5 and NPHP6 gene 3 products. Hum Mol Genet 17(23):3655-62. 4 Seong MW, Kim SY, Yu YS, Hwang JM, Kim JY, Park SS. 2008. Molecular characterization of 5 Leber congenital amaurosis in Koreans. Mol Vis 14:1429-36. 6 Shu X, Black GC, Rice JM, Hart-Holden N, Jones A, O'Grady A, Ramsden S, Wright AF. 2007. 7 RPGR mutation analysis and disease: an update. Hum Mutat 28(4):322-8. 8 Simms RJ, Eley L, Sayer JA. 2009. Nephronophthisis. Eur J Hum Genet 17(4):406-16. 9 Simonelli F, Ziviello C, Testa F, Rossi S, Fazzi E, Bianchi PE, Fossarello M, Signorini S, 10 Bertone C, Galantuomo S, Brancati F, Valente EM, Ciccodicola A, Rinaldi E, Auricchio 11 A, Banfi S. 2007. Clinical and molecular genetics of Leber's congenital amaurosis: a 12 multicenter study of Italian patients. Invest Ophthalmol Vis Sci 48(9):4284-90. 13 Smith UM, Consugar M, Tee LJ, McKee BM, Maina EN, Whelan S, Morgan NV, Goranson E, 14 Gissen P, Lilliquist S, Aligianis IA, Ward CJ, Pasha S, Punyashthiti R, Malik Sharif S, 15 Batman PA, Bennett CP, Woods CG, McKeown C, Bucourt M, Miller CA, Cox P, 16 Algazali L, Trembath RC, Torres VE, Attie-Bitach T, Kelly DA, Maher ER, Gattone VH, 17 2nd, Harris PC, Johnson CA. 2006. The transmembrane protein meckelin (MKS3) is 18 mutated in Meckel-GruberFor syndrome Peer and the wpk rat. NatReview Genet 38(2):191-6. 19 Stone EM. 2003. Finding and interpreting genetic variations that are important to 20 ophthalmologists. Trans Am Ophthalmol Soc 101:437-84. 21 Sundaresan P, Vijayalakshmi P, Thompson S, Ko AC, Fingert JH, Stone EM. 2009. Mutations 22 that are a common cause of Leber congenital amaurosis in northern America are rare in 23 Southern India. Mol Vis 15:1781-7. 24 Tallila J, Jakkula E, Peltonen L, Salonen R, Kestila M. 2008. Identification of CC2D2A as a 25 Meckel syndrome gene adds an important piece to the ciliopathy puzzle. Am J Hum 26 Genet 82(6):1361-7. 27 Tammachote R, Hommerding CJ, Sinders RM, Miller CA, Czarnecki PG, Leightner AC, 28 Salisbury JL, Ward CJ, Torres VE, Gattone VH, 2nd, Harris PC. 2009. Ciliary and 29 centrosomal defects associated with mutation and depletion of the Meckel syndrome 30 genes MKS1 and MKS3. Hum Mol Genet 18(17):3311-23. 31 Thompson S, Whiting RE, Kardon RH, Stone EM, Narfstrom K. 2010. Effects of hereditary 32 retinal degeneration due to a CEP290 mutation on the feline pupillary light reflex. Vet 33 Ophthalmol 13(3):151-7. 34 Torrado M, Iglesias R, Nespereira B, Mikhailov AT. 2010. Identification of candidate genes 35 potentially relevant to chamber-specific remodeling in postnatal ventricular myocardium. 36 J Biomed Biotechnol 2010:603159. 37 Tory K, Lacoste T, Burglen L, Moriniere V, Boddaert N, Macher MA, Llanas B, Nivet H, 38 Bensman A, Niaudet P, Antignac C, Salomon R, Saunier S. 2007. High NPHP1 and 39 NPHP6 mutation rate in patients with Joubert syndrome and nephronophthisis: potential 40 epistatic effect of NPHP6 and AHI1 mutations in patients with NPHP1 mutations. J Am 41 Soc Nephrol 18(5):1566-75. 42 Travaglini L, Brancati F, Attie-Bitach T, Audollent S, Bertini E, Kaplan J, Perrault I, Iannicelli 43 M, Mancuso B, Rigoli L, Rozet JM, Swistun D, Tolentino J, Dallapiccola B, Gleeson JG, 44 Valente EM, Zankl A, Leventer R, Grattan-Smith P, Janecke A, D'Hooghe M, Sznajer Y, 45 Van Coster R, Demerleir L, Dias K, Moco C, Moreira A, Kim CA, Maegawa G, Petkovic 46 D, Abdel-Salam GM, Abdel-Aleem A, Zaki MS, Marti I, Quijano-Roy S, Sigaudy S, de 47 Lonlay P, Romano S, Touraine R, Koenig M, Lagier-Tourenne C, Messer J, Collignon P, 48 Wolf N, Philippi H, Kitsiou Tzeli S, Halldorsson S, Johannsdottir J, Ludvigsson P, 49 34 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 35 of 51 Human Mutation

1 2 Phadke SR, Udani V, Stuart B, Magee A, Lev D, Michelson M, Ben-Zeev B, Fischetto R, 3 Benedicenti F, Stanzial F, Borgatti R, Accorsi P, Battaglia S, Fazzi E, Giordano L, Pinelli 4 L, Boccone L, Bigoni S, Ferlini A, Donati MA, Caridi G, Divizia MT, Faravelli F, 5 Ghiggeri G, Pessagno A, Briguglio M, Briuglia S, Salpietro CD, Tortorella G, Adami A, 6 Castorina P, Lalatta F, Marra G, Riva D, Scelsa B, Spaccini L, Uziel G, Del Giudice E, 7 Laverda AM, Ludwig K, Permunian A, Suppiej A, Signorini S, Uggetti C, Battini R, Di 8 Giacomo M, Cilio MR, Di Sabato ML, Leuzzi V, Parisi P, Pollazzon M, Silengo M, De 9 Vescovi R, Greco D, Romano C, Cazzagon M, Simonati A, Al-Tawari AA, Bastaki L, 10 Megarbane A, Sabolic Avramovska V, de Jong MM, Stromme P, Koul R, Rajab A, Azam 11 M, Barbot C, Martorell Sampol L, Rodriguez B, Pascual-Castroviejo I, Teber S, Anlar B, 12 Comu S, Karaca E, Kayserili H, Yuksel A, Akcakus M, Al Gazali L, Sztriha L, Nicholl D, 13 Woods CG, Bennett C, Hurst J, Sheridan E, Barnicoat A, Hennekam R, Lees M, Blair E, 14 Bernes S, Sanchez H, Clark AE, DeMarco E, Donahue C, Sherr E, Hahn J, Sanger TD, 15 Gallager TE, Dobyns WB, Daugherty C, Krishnamoorthy KS, Sarco D, Walsh CA, 16 McKanna T, Milisa J, Chung WK, De Vivo DC, Raynes H, Schubert R, Seward A, 17 Brooks DG, Goldstein A, Caldwell J, Finsecke E, Maria BL, Holden K, Cruse RP, 18 Swoboda KJ, ViskochilFor D. 2009. Expanding Peer CEP290 mutReviewational spectrum in ciliopathies. 19 Am J Med Genet A 149A(10):2173-80. 20 Tsang WY, Bossard C, Khanna H, Peranen J, Swaroop A, Malhotra V, Dynlacht BD. 2008. 21 CP110 suppresses primary cilia formation through its interaction with CEP290, a protein 22 deficient in human ciliary disease. Dev Cell 15(2):187-97. 23 Valente EM, Brancati F, Dallapiccola B. 2008. Genotypes and phenotypes of Joubert syndrome 24 and related disorders. Eur J Med Genet 51(1):1-23. 25 Valente EM, Logan CV, Mougou-Zerelli S, Lee JH, Silhavy JL, Brancati F, Iannicelli M, 26 Travaglini L, Romani S, Illi B, Adams M, Szymanska K, Mazzotta A, Lee JE, Tolentino 27 JC, Swistun D, Salpietro CD, Fede C, Gabriel S, Russ C, Cibulskis K, Sougnez C, 28 Hildebrandt F, Otto EA, Held S, Diplas BH, Davis EE, Mikula M, Strom CM, Ben-Zeev 29 B, Lev D, Sagie TL, Michelson M, Yaron Y, Krause A, Boltshauser E, Elkhartoufi N, 30 Roume J, Shalev S, Munnich A, Saunier S, Inglehearn C, Saad A, Alkindy A, Thomas S, 31 Vekemans M, Dallapiccola B, Katsanis N, Johnson CA, Attie-Bitach T, Gleeson JG. 32 2010. Mutations in TMEM216 perturb ciliogenesis and cause Joubert, Meckel and related 33 syndromes. Nat Genet. In Press. 34 Valente EM, Silhavy JL, Brancati F, Barrano G, Krishnaswami SR, Castori M, Lancaster MA, 35 Boltshauser E, Boccone L, Al-Gazali L, Fazzi E, Signorini S, Louie CM, Bellacchio E, 36 Bertini E, Dallapiccola B, Gleeson JG. 2006. Mutations in CEP290, which encodes a 37 centrosomal protein, cause pleiotropic forms of Joubert syndrome. Nat Genet 38(6):623-5. 38 Vallespin E, Lopez-Martinez MA, Cantalapiedra D, Riveiro-Alvarez R, Aguirre-Lamban J, 39 Avila-Fernandez A, Villaverde C, Trujillo-Tiebas MJ, Ayuso C. 2007. Frequency of 40 CEP290 c.2991_1655A>G mutation in 175 Spanish families affected with Leber 41 congenital amaurosis and early-onset retinitis pigmentosa. Mol Vis 13:2160-2. 42 Walia S, Fishman GA, Jacobson SG, Aleman TS, Koenekoop RK, Traboulsi EI, Weleber RG, 43 Pennesi ME, Heon E, Drack A, Lam BL, Allikmets R, Stone EM. 2010. Visual Acuity in 44 Patients with Leber's Congenital Amaurosis and Early Childhood-Onset Retinitis 45 Pigmentosa. Ophthalmology 117:1190–1198 . Deleted : . 46 Wang M, Bridges JP, Na CL, Xu Y, Weaver TE. 2009. Meckel-Gruber syndrome protein MKS3 47 is required for endoplasmic reticulum-associated degradation of surfactant protein C. J 48 Biol Chem 284(48):33377-83. 49 35 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 36 of 51

1 2 Zaghloul NA, Katsanis N. 2009. Mechanistic insights into Bardet-Biedl syndrome, a model 3 ciliopathy. J Clin Invest 119(3):428-37. 4 5 6 Deleted : ¶ 7 Figure legends 8 9 10 CEP290 11 Figure 1: Overview of all mutations and variants currently present in base. 12 The exact location of the mutation/variant is depicted with respect to the CEP290 protein (Sayer 13 14 et al., 2006). If no protein effect is known, the c.DNA nomenclature is used. Mutations 15 depicted twice have been reported to arise from different nucleotide changes. 16

17 18 Figure 2: Overlap of CEP290For mutations betweenPeer different Reviewdiseases. 19 Abbreviations used: LCA: Leber Congenital Amaurosis; SLS: Senior-Loken syndrome; JS: 20 21 Joubert syndrome; JS + RD: Joubert syndrome with associated retinopathy; JS + RF: 22 Joubert syndrome with associated renal failure; CORS: cerebello-oculo-renal syndrome; 23 24 MKS: Meckel-Grüber syndrome; MKS-like: Meckel-Grüber syndrome – like; BBS: 25 Bardet-Biedl syndrome. 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 36 50

51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 37 of 51 Human Mutation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 base 24 Figure 1: Overview of all mutations and variants currently present in CEP290 . 25 The exact location of the mutation/variant is depicted with respect to the CEP290 protein (Sayer et al., 2006). If no protein effect is known, the c.DNA nomenclature is used. Mutations depicted twice 26 have been reported to arise from different nucleotide changes. 27 291x141mm (300 x 300 DPI) 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 38 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 Figure 1: Overview of all mutations and variants currently present in CEP290 base . 26 The exact location of the mutation/variant is depicted with respect to the CEP290 protein (Sayer et 27 al., 2006). If no protein effect is known, the c.DNA nomenclature is used. Mutations depicted twice have been reported to arise from different nucleotide changes. 28 291x141mm (300 x 300 DPI) 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 39 of 51 Human Mutation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Figure 2: Overlap of CEP290 mutations between different diseases. 34 Abbreviations used: LCA: Leber Congenital Amaurosis; SLS: Senior-Loken syndrome; JS: Joubert syndrome; JS + RD: Joubert syndrome with associated retinopathy; JS + RF: Joubert syndrome 35 with associated renal failure; CORS: cerebello-oculo-renal syndrome; MKS: Meckel-Grüber 36 syndrome; MKS-like: Meckel-Grüber syndrome – like; BBS: Bardet-Biedl syndrome. 37 165x121mm (300 x 300 DPI) 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 40 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Figure 2: Overlap of CEP290 mutations between different diseases. 34 Abbreviations used: LCA: Leber Congenital Amaurosis; SLS: Senior-Loken syndrome; JS: Joubert syndrome; JS + RD: Joubert syndrome with associated retinopathy; JS + RF: Joubert syndrome 35 with associated renal failure; CORS: cerebello-oculo-renal syndrome; MKS: Meckel-Grüber 36 syndrome; MKS-like: Meckel-Grüber syndrome – like; BBS: Bardet-Biedl syndrome. 37 165x121mm (300 x 300 DPI) 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 41 of 51 Human Mutation

1 Deleted: 3 2 Table 1: Overview of selected ciliary proteins interacting with CEP290, and their 3 4 involvement in partially overlapping yet distinct ciliopathies. 5 6 7 Associated phenotype Protein 8 RD NPHP SLS JS(RD) MKS BBS COACH 9 (Sayer, 10 et al., (den (Helou (Sayer et (Baala et 11 2006; (Leitch et CEP290 Hollander et et al., al., al., Valente al., 2008) 12 al., 2006) 2007)(?) 2006) 2007a) 13 et al., 2006) 14 (Meindl et 15 RPGR 16 al., 1996) (Dryja et al., 17 RPGRIP1 18 2001) For Peer Review 19 (Otto et 20 NPHP5 al., 2005) 21 22 TMEM67 (Baala et (Smith et (Brancati al., al., et al., Deleted: MKS3 ¶ 23 (MKS3 ) 2007b) 2006) 2009) 24 Deleted: TMEM67 CEP290interacating proteins (Gorden (Tallila (Doherty 25 CC2D2A et al., et al., et al., 26 2008) 2008) 2009) 27 Abbreviations used: RD: retinal degeneration; NPHP: nephronophthisis; SLS: Senior-Loken 28 29 syndrome; JS(RD): Joubert syndrome (related disorders); MKS: Meckel-Grüber syndrome; BBS: 30 31 Bardet-Biedl syndrome; COACH: cerebellar vermis hypo/aplasia, oligophrenia, congenital 32 33 ataxia, ocular coloboma, and hepatic fibrosis; (?): uncertain association. 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 42 of 51

1 2 Supp. Figure S1: Overview of CEP290 base 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 43 of 51 Human Mutation

1 2 The database is accessible from: http://medgen.ugent.be/cep290base. Information on variants can 3 4 be retrieved using the Mutation Overview (1) or the Mutation Browser (2). In addition, the 5 6 Phenotype page (3) describes all phenotypes associated with CEP290 so far. The variant-specific 7 8 pages (4) are accessible from both the Mutation Overview and Mutation Browser and include 9 10 detailed information on the pathogenic potential. The database links to NetGene2 and BDGP for 11 12 splice site mutations and automatically fills in queries for PolyPhen and SIFT for missense 13 14 changes. In addition, the database includes variants in other genes that co-occur with CEP290 15 16 mutations, thereby providing a unique opportunity to link modifiers to associated clinical 17 18 manifestations. For Peer Review 19 20 21 22 23

24 25 26 27 28 29 30 31

32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG 47 translation initiation codon in the reference sequence (NM_025114.3), according to journal 48 guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1. Page 2 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 44 of 51

1 Deleted: Table 1 2 Supp. Table S1 : Overview of all CEP290 mutations identified so far and their occurrence in 3 4 different phenotypes. 5 6 7 Mutation Associated phenotype 8 JS JS Nucleotide Protein MKS- 9 LCA SLS JS + + CORS MKS BBS nomenclature nomenclature like 10 RD RF 11 c.1A>G p.Met1? 1 12 c.2T>A p.Met1? 1 13 14 c.21G>T p.Trp7Cys 1 1 15 c.103-13_103-18del p.? 1 16 c.164_167del p.Thr55SerfsX3 1 17 c.180+1G>T p.? 1 18 c.180+2T>A For p.? Peer Review 2 (1) 19 20 c.265dup p.Thr89AsnfsX2 1 21 c.287del p.Asn96MetfsX29 1* 1* 22 c.322C>T p.Arg108X 1 23 c.381_382delinsT p.Lys127AsnfsX36 1 24 c.384_385del p.Asp128GlufsX17 1 25 26 c.384_387del p.Asp128GlufsX34 1 4 (2) 27 c.437del p.Glu146GlyfsX17 1 28 c.451C>T p.Arg151X 1 29 c.566C>G p.Ser189X 1 30 c.613C>T p.Arg205X 2 (1) 31 32 c.679_680del p.Glu227SerfsX2 1 33 c.1066-1G>A p.? 1 34 c.1189+1G>A p.? 1 35 c.1219_1220del p.Met407GlufsX14 3 1 36 c.1260_1264del p.Lys421GlyfsX2 2 37 38 c.1361del p.Gly454GlufsX5 1 39 c.1419_1423del p.Ile474ArgfsX5 1 40 c.1550del p.Leu517X 1 41 c.1593C>A p.Tyr531X 1 42 c.1645C>T p.Arg549X 1 43 44 c.1666del p.Ile556PhefsX17 1 45 46 Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG 47 translation initiation codon in the reference sequence (NM_025114.3), according to journal 48 guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1. Page 3 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 45 of 51 Human Mutation

1 2 Mutation Associated phenotype 3 JS JS 4 Nucleotide Protein MKS- LCA SLS JS + + CORS MKS BBS nomenclature nomenclature like 5 RD RF 6 c.1682_1683del p.Gln561ArgfsX14 1 7 8 c.1709C>G p.Ser570X 1 9 c.1711+5A>G p.? 1 10 c.1824G>A p.= 1 11 c.1859_1862del p.Arg621IlefsX2 2 12 13 c.1860_1861del p.Asp622PhefsX5 1 14 c.1910-2A>C p.? 1 15 c.1936C>T p.Gln646X 2 (1) 16 c.1984C>T p.Gln662X 2 (1) 17 c.1992del p.Pro665LeufsX10 1 18 For Peer Review 19 c.2118_2122dup p.Thr709SerfsX9 1 20 c.2218-15_2220del p.? 1 21 c.2218-4_2222del p.? 2 (1) Deleted: c.2118_2122dup ... [1] 22 c.2218-2A>C p.? 1 23 c.2249T>G p.Leu750X 1 1 24 25 c.2251C>T p.Arg751X 1 26 c.2695C>T p.Gln899X 1 27 c.2906dup p.Tyr969X 1 28 92 c.2991+1655A>G p.Cys998X 29 (83) 30 c.3043G>T p.Glu1015X 1 31 c.3104-2A>G p.? 1 32 33 c.3104-1G>A p.? 1 34 c.3175del p.Ile1059X 1 35 c.3175dup p.Ile1059AsnfsX11 1 1 1 36 c.3176del p.Ile1059LysfsX6 1 37 c.3292G>T p.Glu1098X 2 38 39 c.3310-1G>C p.? 1 c.3310- 40 p.? 3 41 1_3310delinsAA 42 c.3422dup p.Leu1141PhefsX5 1 43 c.3793C>T p.Gln1265X 1 1 44 45 46 Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG 47 translation initiation codon in the reference sequence (NM_025114.3), according to journal 48 guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1. Page 4 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 46 of 51

1 2 Mutation Associated phenotype 3 JS JS 4 Nucleotide Protein MKS- LCA SLS JS + + CORS MKS BBS nomenclature nomenclature like 5 RD RF 6 c.3811C>T p.Arg1271X 1 4 (2) 7 8 c.3814C>T p.Arg1272X 1 1 9 c.3922C>T p.Gln1308X 1 10 c.4001del p.Thr1334IlefsX2 1 11 c.4028del p.Lys1343ArgfsX2 1 12 13 c.4114_4115del p.Ile1372LysfsX5 1 14 c.4115_4116del p.Ile1372LysfsX5 1 1 15 c.4195-1G>A p.? 1 16 c.4393C>T p.Arg1465X 1 2 3 17 c.4656del p.Glu1553LysfsX4 1 18 For Peer Review 19 c.4723A>T p.Lys1575X 12 2 1 1 3 20 c.4732G>T p.Glu1578X 21 (1) 22 c.4771C>T p.Gln1591X 2 (1) 23 c.4791_4794del p.Lys1598SerfsX8 1 24 c.4882C>T p.Gln1628X 2 1* 3* 25 c.4962_4963del p.Glu1656AsnfsX3 2 26 27 c.4965_4966del p.Glu1656AsnfsX3 1 28 c.4966G>T p.Glu1656X 1 29 c.5046del p.Val1683X 1 30 c.5163del p.Thr1722GlnfsX2 3 2 31 c.5226+1G>A p.? 1 32 33 c.5256_5257del p.Ala1753ArgfsX7 2 (1) 34 c.5344C>T p.Arg1782X 1 35 c.5434_5435del p.Glu1812LysfsX5 1 36 c.5493del p.Ala1832ProfsX19 1 1 4 (2) 37 c.5515_5518del p.Glu1839LysfsX11 1 38 39 c.5519_5537del p.Lys1840ArgfsX5 1 40 c.5587-1G>C p.? 3 2 41 c.5611_5614del p.Gln1871ValfsX2 1* 1* 42 c.5649dup p.Leu1884ThrfsX23 1* 1* 3 (2) 2 (1) 43 c.5668G>T p.Gly1890X 2 (1) 1 9 (7) 44 3 45 46 Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG 47 translation initiation codon in the reference sequence (NM_025114.3), according to journal 48 guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1. Page 5 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 47 of 51 Human Mutation

1 2 Mutation Associated phenotype 3 JS JS 4 Nucleotide Protein MKS- LCA SLS JS + + CORS MKS BBS nomenclature nomenclature like 5 RD RF 6 (2) 7 8 ¥ p.Glu1903X 1 9 CEP290:c.5709+2352_ 54_ 10 p.? 1 11 C12orf50:c.290– 1375_77del 12 13 c.5722G>T p.Glu1908X 1 14 c.5734del p.Trp1912GlyfsX11 4 (2) 15 c.5776C>T p.Arg1926X 1 16 c.5813_5817del p.Thr1938AsnfsX16 1 17 c.5824C>T p.Gln1942X 1* 1* 18 For Peer Review 19 c.5850del p.Phe1950LeufsX15 3 2 (1) 2 (1) 20 c.5865_5867delinsGG p.Glu1956GlyfsX9 1 21 c.5866G>T p.Glu1956X 1 22 c.5932C>T p.Arg1978X 1 23 24 c.5941G>T p.Glu1981X 2 25 c.6031C>T p.Arg2011X 1 26 c.6072C>A p.Tyr2024X 2 (1) 27 c.6271-8T>G p.? 1 28 c.6277del p.Val2093SerfsX4 1 29 30 c.6331C>T p.Gln2111X 1 31 c.6604del p.Ile2202LeufsX24 7 (2) 32 c.6869del p.Asn2290IlefsX11 2 33 c.6870del p.Gln2291LysfsX10 2 (1) 34 c.7318_7321dup p.Leu2441SerfsX16 2 (1) 35 Deleted: 1 36 c.7341dup p.Leu2448ThrfsX8 1 2 2 37 c.7366_7369del p.Thr2457AlafsX27 38 (1) 23 Deleted: 22 39 184 13 4 65 17 10 Total number of distinct patients (families) 2 (20 1 40 (167) (12) (3) (53) (11) (7) Deleted: 19 41 ) 42 This table gives an overview of the number of patients carrying a specific mutation, for each 43 th 44 phenotype (updating date: 14 of June, 2010) . Numbers between brackets indicate the unique 45 46 Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG 47 translation initiation codon in the reference sequence (NM_025114.3), according to journal 48 guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1. Page 6 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 48 of 51

1 2 number of families. Abbreviations used: LCA: Leber Congenital Amaurosis; SLS: Senior-Loken 3 4 syndrome; JS: Joubert syndrome; JS + RD: Joubert syndrome with associated retinopathy; JS + 5 6 RF: Joubert syndrome with associated renal failure; CORS: cerebello-oculo-renal syndrome; 7 Deleted: 8 MKS: Meckel-Grüber syndrome; MKS-like: Meckel-Grüber syndrome -like; BBS: Bardet-Biedl Deleted: – 9 10 syndrome. ¥: nucleotide nomenclature not specified in publication. *: both patients carrying the 11 12 mutation belong to the same family. 13 14 15 16 17

18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG 47 translation initiation codon in the reference sequence (NM_025114.3), according to journal 48 guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1. Page 7 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 49 of 51 Human Mutation

1 Deleted: Table 2 2 Supp. Table S2 : Overview of all CEP290 unclassified variants identified so far and their 3 4 occurrence in different phenotypes. 5 6 7 Unclassified variant Associated phenotype 8 Nucleotide Protein LCA NPHP JS +RF Healthy 9 nomenclature nomenclature 10 c.1991A>G p.Asp664Gly 1 1 11 12 c.2717T>G p.Leu906Trp 1 13 c.6684T>G p.Asn2228Lys 1 14 c.4696G>C p.Ala1566Pro 1 15 c.5081T>C p.Leu1694Pro 1 16 c.4661_4663del p.Glu1554del 1 17 18 c.7311_7313del p.Lys2437delFor Peer Review 1 19 Abbreviations used: LCA: Leber Congenital Amaurosis; NPHP: nephronophthisis; JS + RF: 20 21 Joubert syndrome with associated renal failure. 22 23

24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG 47 translation initiation codon in the reference sequence (NM_025114.3), according to journal 48 guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1. Page 8 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 50 of 51

1 2 3 Page 4: [1] Deleted Frauke Coppieters 6/14/2010 5:55:00 PM 4 c.2118_2122dup p.Thr709SerfsX9 1 5

6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 51 of 51 Human Mutation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 Supp. Figure S1: Overview of CEP290 base . 20 The database is accessible from: http://medgen.ugent.be/cep290base. Information on variants can 21 be retrieved using the Mutation Overview (1) or the Mutation Browser (2). In addition, the 22 Phenotype page (3) describes all phenotypes associated with CEP290 so far. The variant-specific 23 pages (4) are accessible from both the Mutation Overview and Mutation Browser and include 24 detailed information on the pathogenic potential. The database links to NetGene2 and BDGP for 25 splice site mutations and automatically fills in queries for PolyPhen and SIFT for missense changes. In addition, the database includes variants in other genes that co-occur with CEP290 mutations, 26 thereby providing a unique opportunity to link modifiers to associated clinical manifestations. 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc.