Frauke ; Coppieters

Steve Lefever

Elfride Bart P Leroy

Bw De Baere

MD Elfride De Baere

PhD Elfride Debaere

Jo Vandesompele

CEP290, a gene with many faces: mutation overview and presentation of CEP290base

Keywords: CEP290, locus-specific database, genotype-phenotype correlations, modifiers, ciliary proteome CEP290, locus-specific database, genotype-phenotype correlations, modifiers, ciliary proteome _382delinsT, c.384_385del and c.384_387del

come

Background

Cilia are highly conserved organelles that are essential for many cell types. Apart from their obvious role in motility and transport of fluids and particles over epithelial surfaces, they have numerous other functions such as signal transduction [START_REF] Berbari | The primary cilium as a complex signaling center[END_REF]. The extensive presence of cilia throughout the whole body might explain the wide range of phenotypes associated with mutations in genes encoding ciliary proteins [START_REF] Gerdes | The vertebrate primary cilium in development, homeostasis, and disease[END_REF][START_REF] Nigg | Centrioles, centrosomes, and cilia in health and disease[END_REF].

One of the most intriguing disease genes associated with ciliopathies is CEP290 (MIM [610142]), as the phenotypic spectrum of its mutations ranges from isolated blindness to the lethal Meckel-Grüber syndrome (MKS). The gene was initially identified as disease gene for Joubert syndrome (related disorders) (JS [RD]) and Senior-Loken syndrome (SLS) [START_REF] Sayer | The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4[END_REF][START_REF] Valente | Mutations in CEP290, which encodes a centrosomal protein, cause pleiotropic forms of Joubert syndrome[END_REF]. Within a few years, Leber Congenital Amaurosis (LCA), MKS and Bardet-Biedl syndrome (BBS) expanded the list of partially overlapping yet distinct disorders caused by CEP290 mutations (den [START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Under review[END_REF]Baala et al., 2007a;[START_REF] Leitch | Hypomorphic mutations in syndromic encephalocele genes are associated with Bardet-Biedl syndrome[END_REF]. Although these are essentially autosomal recessive (AR) monogenic diseases, epistatic effects of modifier alleles in additional ciliary genes should not be underestimated in the development of their phenotypes.

The first clone corresponding with CEP290, KIAA0373, was identified through sequencing of 100 new cDNA clones from human brain cDNA libraries [START_REF] Nagase | Prediction of the coding sequences of unidentified human genes. VII. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro[END_REF]. Three years later, Chen and Shou independently cloned CEP290 as 3H11Ag, encoding an antigen for the monoclonal antibody 3H11 which specifically recognizes cancer cells from various tissues [START_REF] Chen | Molecular cloning of a tumor-associated antigen recognized by monoclonal antibody 3H11[END_REF]. It was predicted that about 60% of the residues form coiled-coil (CC) structures novel AAs (c.1550del, c.2906dup, c.3175del andc.5046del). Taking into account the position of the PTC relative to the ultimate 3′ exon-exon junction, 85 truncating mutations are assumed to undergo nonsense mediated decay (NMD) [START_REF] Nagy | A rule for termination-codon position within intron-containing genes: when nonsense affects RNA abundance[END_REF]. However, the p.Arg151X mutation was shown to result in alternative transcripts, lacking exon 7 or both exon 7 and 8, leaving the open reading frame intact [START_REF] Littink | A novel nonsense mutation in CEP290 induces exon skipping and leads to a relatively mild retinal phenotype[END_REF]. Three truncating mutations located in the last exon are expected to escape NMD and render a protein product (c.7318_7321dup, c.7341dup and c.7366_7369del).

In addition to the 88 truncating mutations caused by small variations, a large heterozygous deletion was recently identified in a patient with cerebello-oculo-renal syndrome (CORS). The deleted region spanned 76,844 bp at the genomic level, encompassing the last 13 coding exons of CEP290, the entire C12orf29 gene and part of the C12orf50 gene (CEP290:c.5709+2352_54_C12orf50:c.290-1375_77del) (Travaglini et al., 2009).

The remaining 23 mutations comprise 3 missense mutations and 20 mutations predicted to influence splicing. Of the latter, 12 mutations affect consensus donor or acceptor splice sites, whereas three mutations are located within 20 nucleotides surrounding the exon. The first two, c.103-13_103-18del and c.6271-8T>G, were considered to be likely pathogenic because they decreased the scores of the normal splice sites and because they are absent in more than 115 control individuals [START_REF] Tory | High NPHP1 and NPHP6 mutation rate in patients with Joubert syndrome and nephronophthisis: potential epistatic effect of NPHP6 and AHI1 mutations in patients with NPHP1 mutations[END_REF]. The third one, c.1711+5A>G, was presumed to result in both abnormally and normally spliced transcripts [START_REF] Perrault | Spectrum of NPHP6/CEP290 mutations in Leber congenital amaurosis and delineation of the associated phenotype[END_REF]. Splice site prediction scores, however, remain unchanged for this variant (Alamut v.1.5, data not shown). In addition, an aberrant splicing pattern was predicted for the c.1824G>A mutation affecting the last nucleotide of exon 18 [START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Under review[END_REF]. Surprisingly, the most recurrent mutation, c.2991+1655A>G, represents a deep intronic mutation. It creates a strong splice donor site that results in the insertion of a 128-bp cryptic exon between exons 26 and 27, thereby leading to a PTC immediately downstream of exon 26 (p.Cys998X) (den [START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Under review[END_REF]. Apart from these 17 substitutions, two deletions and one indel overspan an intron-exon boundary (c.2218(c. -4_2222del, c.2218-15_2220del and c.3310-1_3310delinsAA)-15_2220del and c.3310-1_3310delinsAA).

So far, only three missense variants have been described with a probable pathogenic effect (see "Polymorphisms and unclassified variants" for other missense variants). Two affect the start codon (c.1A>G and c.2T>A) whereas the third, p.Trp7Cys, affects a highly conserved AA and is predicted to disrupt protein function according to SIFT, PolyPhen and Grantham matrix.

However, the mutant protein was correctly localized at centrosomes in mouse inner medullary collecting duct (IMCD-3) cells, suggesting a pathogenic mechanism different from mislocalization [START_REF] Valente | Mutations in CEP290, which encodes a centrosomal protein, cause pleiotropic forms of Joubert syndrome[END_REF].

Interestingly, some mutations cluster in the same region or even codon, suggesting the existence of mutation hotspots. In nine coding regions, two or more mutations arose either with a different effect on protein level (start codon; c.381-387; c.1859-1862; c.3175-3176; c.4965-4966; c.5515-5537; c.5865-5867; c.6869-6870) or with an identical predicted protein effect (c.4114-4116; c.4962-4966). In addition, one donor and three acceptor splice sites displayed more than one mutation (c. 180+1G>T and c.180+2T>A;c.2218-2A>C, c.2218-4_2222del and c.2218-15_2220del;c.3104-1G>A and c.3104-2A>G;c.3310-1_3310delinsAA and c.3310-1G>C). In general, mutations are scattered throughout the protein, with some clustering in CCs III, XI and XII (Figure 1). Coiled-coils might be involved in the overall conformation of CEP290. Therefore, mutations in them might affect the accessibility of interacting proteins [START_REF] Schafer | Genetic and physical interaction between the NPHP5 and NPHP6 gene products[END_REF]. A total of 83 mutations are unique whereas 26 mutations have been reported in less than 10 families (Supp. Table S1). A few other mutations occurred multiple times and might even represent founder mutations. The most recurrent one is c.2991+1655A>G. In northwestern Europe, this mutation occurs in up to 26% of all LCA cases (den [START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Under review[END_REF][START_REF] Perrault | Spectrum of NPHP6/CEP290 mutations in Leber congenital amaurosis and delineation of the associated phenotype[END_REF][START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Under review[END_REF]. In Southern Europe, Korea, Southern India and Saudi-Arabia, however, c.2991+1655A>G has a significantly lower prevalence [START_REF] Simonelli | Clinical and molecular genetics of Leber's congenital amaurosis: a multicenter study of Italian patients[END_REF][START_REF] Vallespin | Frequency of CEP290 c.2991_1655A>G mutation in 175 Spanish families affected with Leber congenital amaurosis and early-onset retinitis pigmentosa[END_REF][START_REF] Seong | Molecular characterization of Leber congenital amaurosis in Koreans[END_REF][START_REF] Li | Mutation survey of known LCA genes and loci in the Saudi Arabian population[END_REF][START_REF] Sundaresan | Mutations that are a common cause of Leber congenital amaurosis in northern America are rare in Southern India[END_REF]. The secondmost frequent mutation, p.Lys1575X, was so far only reported in probands originating from France (Lille) and northern Belgium [START_REF] Brancati | CEP290 mutations are frequently identified in the oculo-renal form of Joubert syndrome-related disorders[END_REF][START_REF] Perrault | Spectrum of NPHP6/CEP290 mutations in Leber congenital amaurosis and delineation of the associated phenotype[END_REF][START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Under review[END_REF]. Haplotype analysis in seven non-consanguineous families revealed a common intragenic allele but distinct extragenic haplotypes, suggesting an ancient mutation [START_REF] Perrault | Spectrum of NPHP6/CEP290 mutations in Leber congenital amaurosis and delineation of the associated phenotype[END_REF].

For p.Ala1832ProfsX19, allele sharing analysis in two families with MKS originating from Kosovo and Kosovo-Albania was in favor of a common founder haplotype encompassing approximately 3 Mb [START_REF] Frank | Mutations of the CEP290 gene encoding a centrosomal protein cause Meckel-Gruber syndrome[END_REF]. In contrast, different haplotypes in two MKS families of Tunisian and French origin point to a mutation hotspot for p.Asp128GlufsX34 (Baala et al., 2007a). All mutations segregated in the parents (as far as this could be assessed), with the exception of p.Gln2111X, which was reported to arise de novo in a patient with CORS (Sayer et al., 2006).

In 18 patients, only one mutation was identified. This might be due to an inability of standard Similar arguments could also apply for five families with isolated NPHP in which no mutations could be identified at all, despite linkage to a region containing CEP290 [START_REF] Helou | Mutation analysis of NPHP6/CEP290 in patients with Joubert syndrome and Senior-Loken syndrome[END_REF].

Alternatively, another as yet unidentified NPHP gene could segregate in linkage disequilibrium with CEP290 in these families.

Polymorphisms and unclassified variants

Several polymorphisms have been described in CEP290, for which we refer to dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP) and Brancati and colleagues [START_REF] Brancati | CEP290 mutations are frequently identified in the oculo-renal form of Joubert syndrome-related disorders[END_REF].

They were predicted not to alter splicing patterns or to impair protein function, and/or were present in over 2% of parents of affected individuals [START_REF] Brancati | CEP290 mutations are frequently identified in the oculo-renal form of Joubert syndrome-related disorders[END_REF]. In addition, the pathogenic potential of some variants is indefinite (Supp. Table S2). Indeed, for several missense variants, in silico predictions are not conclusive (PolyPhen, SIFT and Grantham matrix). The first one is p.Asp664Gly. Despite the identification of this variant in a heterozygous state in a patient with JS and renal involvement, p.Asp664Gly also occurred in a healthy parent of another family, but not in his affected child [START_REF] Brancati | CEP290 mutations are frequently identified in the oculo-renal form of Joubert syndrome-related disorders[END_REF][START_REF] Helou | Mutation analysis of NPHP6/CEP290 in patients with Joubert syndrome and Senior-Loken syndrome[END_REF]. Interestingly, Tory and colleagues screened this variant together with three other CEP290 missense variants (p.Glu277Gln, p.Lys838Glu and p.Arg1746Gln) in the context of modifier identification in the following three cohorts: 1) 13 patients carrying NPHP1 mutation(s) with neurological symptoms;

2) 77-82 patients carrying NPHP1 mutation(s) without neurological symptoms and 3) 132-154 healthy control subjects. No significant difference in frequencies was found among these three cohorts, however [START_REF] Tory | High NPHP1 and NPHP6 mutation rate in patients with Joubert syndrome and nephronophthisis: potential epistatic effect of NPHP6 and AHI1 mutations in patients with NPHP1 mutations[END_REF]. Other missense variants of unknown significance are p.Leu906Trp, p.Asn2228Lys, p.Ala1566Pro and p.Leu1694Pro [START_REF] Brancati | CEP290 mutations are frequently identified in the oculo-renal form of Joubert syndrome-related disorders[END_REF][START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Under review[END_REF]. The latter two were found in two LCA patients, in compound heterozygosity with a nonsense and splice site mutation, respectively, and segregated in healthy parents (Coppieters et Deleted: These

Deleted: Again

Deleted: in Deleted: [START_REF] Perrault | Spectrum of NPHP6/CEP290 mutations in Leber congenital amaurosis and delineation of the associated phenotype[END_REF], while a heterozygous p.Lys2437del variant was identified in a patient with isolated NPHP originating from consanguineous parents [START_REF] Helou | Mutation analysis of NPHP6/CEP290 in patients with Joubert syndrome and Senior-Loken syndrome[END_REF] (Supp. Table S2).

Mutation database

To provide a clear overview of all mutations and variants identified so far in CEP290, we developed CEP290base (medgen.ugent.be/cep290base). This locus-specific mutation database uses a novel scheme in which both genomic and phenotypic data are available, providing the possibility to link patients and their phenotypes to detailed variant information, and vice versa.

Information on variants can be retrieved using the Overview page or the Mutation Browser.

The Overview page displays all unique variants following HGVS guidelines (http://www.hgvs.org/mutnomen) (NM_025114.3), with their occurrence and links to dbSNP, UniProt (http://www.uniprot.org) and OMIM (http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim). In addition, a scaled graphical representation of variants in the CEP290 protein is shown. Variants in the Overview or image are linked to their variant-specific page which, if available, includes the protein domain [START_REF] Sayer | The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4[END_REF], as well as the frequency in control individuals, the RNA effect and the estimate of pathogenic probability [START_REF] Stone | Finding and interpreting genetic variations that are important to ophthalmologists[END_REF]. For truncating mutations, it is indicated whether mRNA might be subjected to NMD [START_REF] Nagy | A rule for termination-codon position within intron-containing genes: when nonsense affects RNA abundance[END_REF], and for missense mutations, the Grantham score is provided [START_REF] Grantham | Amino acid difference formula to help explain protein evolution[END_REF]. The database also links to SIFT (http://sift.jcvi.org) and

PolyPhen (http://genetics.bwh.harvard.edu/pph) prediction servers, using the CEP290 GI number (109255233) and UniProt ID (O15078) for automated input, respectively. Links to NetGene2

Deleted:

Biological relevance

Expression

The original KIAA0373 mRNA was found in kidney and ovary, and to a lesser extent in human thymus, prostate and testis tissue [START_REF] Nagase | Prediction of the coding sequences of unidentified human genes. VII. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro[END_REF]. In addition, it was present in centrosomes from a human lymphoblastoid cell line (KE-37) [START_REF] Andersen | Proteomic characterization of the human centrosome by protein correlation profiling[END_REF]. The independently cloned 3H11Ag mRNA was extensively distributed in embryonic tissue and in different human cancerous tissues, but not in corresponding normal human tissues [START_REF] Chen | Molecular cloning of a tumor-associated antigen recognized by monoclonal antibody 3H11[END_REF]. The 150 C-terminal AAs of 3H11Ag appeared to be responsible for nuclear translocation. In addition, cytoplasmic presence was also established [START_REF] Guo | Subcellullar localization of tumor-associated antigen 3H11Ag[END_REF]. Consistent with the localization of the KIAA0373 protein, CEP290 was found at centrosomes of both ciliated and non-ciliated cells [START_REF] Chang | In-frame deletion in a novel centrosomal/ciliary protein CEP290/NPHP6 perturbs its interaction with RPGR and results in early-onset retinal degeneration in the rd16 mouse[END_REF][START_REF] Sayer | The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4[END_REF][START_REF] Valente | Mutations in CEP290, which encodes a centrosomal protein, cause pleiotropic forms of Joubert syndrome[END_REF]. This centrosomal localization of CEP290 is dynamic throughout the cell cycle, with redistribution to the cytosol starting in prometaphase [START_REF] Sayer | The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4[END_REF]. During the interphase, two or four prominent spots were observed in the G1 and G2 phase. In the G0 phase, CEP290 was found on both the daughter and mother centriole, the latter from which the primary cilium is assembled [START_REF] Tsang | CP110 suppresses primary cilia formation through its interaction with CEP290, a protein deficient in human ciliary disease[END_REF].

Although the centrosomal localization is microtubule-and dynein-independent [START_REF] Chang | In-frame deletion in a novel centrosomal/ciliary protein CEP290/NPHP6 perturbs its interaction with RPGR and results in early-onset retinal degeneration in the rd16 mouse[END_REF][START_REF] Sayer | The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4[END_REF], a major fraction of CEP290 is recruited to centriolar satellites along polymerized microtubules [START_REF] Kim | CEP290 interacts with the centriolar satellite component PCM-1 and is required for Rab8 localization to the primary cilium[END_REF].

In the rod-dominated mouse retina, CEP290 was detected in the connecting cilia, and to a lesser extent in the inner segments [START_REF] Chang | In-frame deletion in a novel centrosomal/ciliary protein CEP290/NPHP6 perturbs its interaction with RPGR and results in early-onset retinal degeneration in the rd16 mouse[END_REF][START_REF] Sayer | The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4[END_REF]. Expression was also established in primate cone photoreceptors [START_REF] Cideciyan | Centrosomal-ciliary gene CEP290/NPHP6 mutations result in blindness with unexpected sparing of photoreceptors and visual brain: implications for therapy of Leber congenital amaurosis[END_REF]. In olfactory sensory neurons, CEP290 localizes to dendritic knobs [START_REF] Mcewen | Hypomorphic CEP290/NPHP6 mutations result in anosmia caused by the selective loss of G proteins in cilia of olfactory sensory neurons[END_REF]. A recent study on differential gene expression in the ventricular myocardium of newborn piglets identified CEP290 as being three- fold more enriched in the right compared with the left ventricle [START_REF] Torrado | Identification of candidate genes potentially relevant to chamber-specific remodeling in postnatal ventricular myocardium[END_REF]. Knockdown experiments in zebrafish using morpholinos caused defects reminiscent of JS, comprising retinal, cerebellar and otic cavity developmental abnormalities as well as pronephric cyst formation, ectopic brain tissue in the fourth ventricle and an abnormal mid-to-hindbrain region associated with hydrocephalus [START_REF] Sayer | The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4[END_REF][START_REF] Schafer | Genetic and physical interaction between the NPHP5 and NPHP6 gene products[END_REF]. Alternatively spliced transcripts/isoforms were identified by us in lymphocytes (data not shown) and observed as bands of low molecular mass on immunoblot analysis in bovine retinal extracts [START_REF] Chang | In-frame deletion in a novel centrosomal/ciliary protein CEP290/NPHP6 perturbs its interaction with RPGR and results in early-onset retinal degeneration in the rd16 mouse[END_REF].

Interaction with other (ciliary) proteins and function

Using a yeast two-hybrid screen and co-immunoprecipitation, Sayer and colleagues identified an interaction between the N-terminal third of CEP290 (exons 2-21) and the C-terminal two thirds of the Activation Transcription Factor 4 (ATF4) protein. Moreover, CEP290 was able to activate ATF4-mediated transcription [START_REF] Sayer | The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4[END_REF]. ATF4 has a protective function by regulating the adaptation of cells to metabolic and oxidative stress, and is required for skeletal and lens development, and haematopoiesis [START_REF] Ameri | Activating transcription factor 4[END_REF]. In addition, association with G γ13 and G olf was identified in mouse olfactory epithelial tissue, suggesting a role of CEP290 in G protein trafficking during olfactory perception [START_REF] Mcewen | Hypomorphic CEP290/NPHP6 mutations result in anosmia caused by the selective loss of G proteins in cilia of olfactory sensory neurons[END_REF].

Moreover, CEP290 interacts with several centrosomal and ciliary proteins. Coimmunoprecipitation experiments using mouse or bovine retinal extracts showed that CEP290 is in complex with dynactin subunits p150 Glued and p50-dynamitin, kinesin subunit KIF3A, kinesinassociated protein (KAP3), the pericentriolar components γ-tubulin and pericentrin, the centriolar marker centrin, PCM1, ninein, SMC1, SMC3, retinitis pigmentosa GTPase regulator (RPGR ORF15 ) and RPGR-interacting protein 1 (RPGRIP1), but not with nucleophosmin, NPHP5 or RP1 [START_REF] Chang | In-frame deletion in a novel centrosomal/ciliary protein CEP290/NPHP6 perturbs its interaction with RPGR and results in early-onset retinal degeneration in the rd16 mouse[END_REF]. In mouse olfactory epithelial tissue, association with p150 Glued , KIF3A, RPGR ORF15 and γ-tubulin, but not BBS4 and IFT88, was also observed [START_REF] Mcewen | Hypomorphic CEP290/NPHP6 mutations result in anosmia caused by the selective loss of G proteins in cilia of olfactory sensory neurons[END_REF]. Of note, a subsequent study showed that pericentrin, γ-tubulin and centrin do not exactly co-localize with CEP290 [START_REF] Kim | CEP290 interacts with the centriolar satellite component PCM-1 and is required for Rab8 localization to the primary cilium[END_REF].

Recently, a potential role for CEP290 in primary cilium assembly was established. Knock-down of CEP290 using siRNAs in human retinal pigment epithelial cells caused a dramatic alteration in the ability of cycling cells to assemble primary cilia on the one hand, and a disrupted migration of mother centrioles to the cell cortex and primary cilia loss in quiescent cells on the other hand [START_REF] Kim | CEP290 interacts with the centriolar satellite component PCM-1 and is required for Rab8 localization to the primary cilium[END_REF][START_REF] Tsang | CP110 suppresses primary cilia formation through its interaction with CEP290, a protein deficient in human ciliary disease[END_REF]. This observation might -at least partially -be attributed to the association of CEP290 with two proteins. First, CEP290 was found to be recruited to centriolar satellites by PCM-1, which is required for the organization of the cytoplasmic microtubule network. Both depletion and overexpression of CEP290 results in redistribution of PCM-1, thereby possibly influencing the transport function of PCM-1 granules between the cytosol and centrosome [START_REF] Kim | CEP290 interacts with the centriolar satellite component PCM-1 and is required for Rab8 localization to the primary cilium[END_REF]. In addition, CEP290 recruits Rab8a, a small GTPase required for ciliary membrane elongation, at centrosomes and cilia [START_REF] Kim | CEP290 interacts with the centriolar satellite component PCM-1 and is required for Rab8 localization to the primary cilium[END_REF][START_REF] Tsang | CP110 suppresses primary cilia formation through its interaction with CEP290, a protein deficient in human ciliary disease[END_REF]. The interaction with Rab8a requires the CEP290 AAs 1208-1695 [START_REF] Tsang | CP110 suppresses primary cilia formation through its interaction with CEP290, a protein deficient in human ciliary disease[END_REF]. In growing cells (not yet capable of ciliogenesis), the latter function of CEP290 is probably inhibited through an interaction with CP110, which prevents CEP290-dependent Rab8a ciliogenesis [START_REF] Tsang | CP110 suppresses primary cilia formation through its interaction with CEP290, a protein deficient in human ciliary disease[END_REF]. Truncating CEP290 mutants defined the following aminoterminal regions necessary for and sufficient to bind CP110: AAs 1-366, AAs 221-366 and AAs 362-822. The binding region of CP110 consists of AAs 1-223, with major involvement of AAs 67-82 [START_REF] Tsang | CP110 suppresses primary cilia formation through its interaction with CEP290, a protein deficient in human ciliary disease[END_REF]. Interestingly, several other CEP290-interacting proteins are also associated with ciliopathies (Table 1). A first one is RPGR. Although a yeast two-hybrid screen could not identify a direct interaction with CEP290, co-immunoprecipitation assays suggested complex formation and both proteins co-localized in IMCD-3 cells and dissociated mouse rods [START_REF] Chang | In-frame deletion in a novel centrosomal/ciliary protein CEP290/NPHP6 perturbs its interaction with RPGR and results in early-onset retinal degeneration in the rd16 mouse[END_REF]. Mutations in RPGR account for approximately 70-90% of X-linked retinitis pigmentosa (RP) and mainly occur in isoforms containing the carboxy-terminal exon open reading frame 15 (RPGR ORF15 ), which are highly expressed in connecting cilia of photoreceptors [START_REF] Hong | RPGR isoforms in photoreceptor connecting cilia and the transitional zone of motile cilia[END_REF][START_REF] Shu | RPGR mutation analysis and disease: an update[END_REF]. In the rd16 mouse (see "Animal models"), mutant CEP290 binds RPGR ORF15 more avidly, leading to aggregation of RPGR ORF15 in the inner segments and redistribution of rhodopsin and arrestin throughout the plasma membrane [START_REF] Chang | In-frame deletion in a novel centrosomal/ciliary protein CEP290/NPHP6 perturbs its interaction with RPGR and results in early-onset retinal degeneration in the rd16 mouse[END_REF].

A second example of a CEP290-interacting protein also associated with ciliary disease is NPHP5, which is associated with SLS. The protein binds calmodulin and is in complex with RPGR ORF15 [START_REF] Otto | Nephrocystin-5, a ciliary IQ domain protein, is mutated in Senior-Loken syndrome and interacts with RPGR and calmodulin[END_REF]. Despite the absence of a complex between CEP290 and NPHP5 in retinal extracts [START_REF] Chang | In-frame deletion in a novel centrosomal/ciliary protein CEP290/NPHP6 perturbs its interaction with RPGR and results in early-onset retinal degeneration in the rd16 mouse[END_REF], NPHP5 was shown to specifically bind a region of CEP290

encompassing CCIII and part of the SMC homology domain (AAs 696 to 896) [START_REF] Schafer | Genetic and physical interaction between the NPHP5 and NPHP6 gene products[END_REF]. Combined knock-down of both proteins in zebrafish embryos synergistically augmented phenotypes seen in embryos treated with morpholinos for either NPHP5 or CEP290. In Xenopus laevis, expression of the NPHP5-binding domain of CEP290 caused substantial neural tube closure defects that were similar to the effect of NPHP5 knock-down. Moreover, co-expression of the NPHP5-binding domain of CEP290 with NPHP5 itself rescued the phenotype, supporting a physical interaction between both proteins in vivo [START_REF] Schafer | Genetic and physical interaction between the NPHP5 and NPHP6 gene products[END_REF].

Both TMEM67 (MKS3) and CC2D2A are also CEP290-interacting proteins involved in disease.

Mutations in both genes may cause MKS, JS and COACH syndrome, which are all severe ciliopathies with multi-systemic involvement [START_REF] Smith | The transmembrane protein meckelin (MKS3) is mutated in Meckel-Gruber syndrome and the wpk rat[END_REF]Baala et al., 2007b;[START_REF] Gorden | CC2D2A is mutated in Joubert syndrome and interacts with the ciliopathy-associated basal body protein CEP290[END_REF][START_REF] Tallila | Identification of CC2D2A as a Meckel syndrome gene adds an important piece to the ciliopathy puzzle[END_REF][START_REF] Brancati | MKS3/TMEM67 mutations are a major cause of COACH Syndrome, a Joubert Syndrome related disorder with liver involvement[END_REF][START_REF] Doherty | Mutations in 3 genes (MKS3, CC2D2A and RPGRIP1L) cause COACH syndrome (Joubert syndrome with congenital hepatic fibrosis)[END_REF]. TMEM67 is involved in both ciliary structure/function and endoplasmic reticulum-associated degradation of surfactant protein C [START_REF] Tammachote | Ciliary and centrosomal defects associated with mutation and depletion of the Meckel syndrome genes MKS1 and MKS3[END_REF][START_REF] Wang | Meckel-Gruber syndrome protein MKS3 is required for endoplasmic reticulum-associated degradation of surfactant protein C[END_REF]. Morpholino experiments in zebrafish revealed a strong genetic interaction between cep290 and Tmem67, with more severe phenotypic effects following knock-down of both genes, compared with depletion of only one of both [START_REF] Leitch | Hypomorphic mutations in syndromic encephalocele genes are associated with Bardet-Biedl syndrome[END_REF]. CC2D2A is presumed to act as a sensor for intracellular calcium and is part of the basal body complex from which the cilium is assembled, where it co-localizes with CEP290 [START_REF] Gorden | CC2D2A is mutated in Joubert syndrome and interacts with the ciliopathy-associated basal body protein CEP290[END_REF][START_REF] Tallila | Identification of CC2D2A as a Meckel syndrome gene adds an important piece to the ciliopathy puzzle[END_REF]. The interaction between both proteins involves the 998 N-terminal AAs of CC2D2A and a fragment of CEP290 containing CCs IV-VI (AAs 703-1130).

A functional interaction was observed in the pronephros of sentinel zebrafish [START_REF] Gorden | CC2D2A is mutated in Joubert syndrome and interacts with the ciliopathy-associated basal body protein CEP290[END_REF].

The involvement of CEP290 in the above-mentioned ciliary complexes strongly supports a role in ciliogenesis and ciliary trafficking. The (tissue-specific) access of its binding partners is possibly regulated by conformational changes of CEP290, since both the N-terminal (AAs 1-695) and Cterminal (AAs 1966-2479) domains are involved in homo-and heterodimeric interactions [START_REF] Schafer | Genetic and physical interaction between the NPHP5 and NPHP6 gene products[END_REF].

Animal models

So far, two naturally occurring animal models are known with mutations in cep290: a pedigree of [START_REF] Thompson | Effects of hereditary retinal degeneration due to a CEP290 mutation on the feline pupillary light reflex[END_REF][START_REF] Narfstrom | Retinal degeneration in the Abyssinian and Somali cat (rdAc): correlation between genotype and phenotype and rdAc allele frequency in two continents[END_REF]. Vacuolization and degeneration of membranes in the basal part of the rod outer segments (OS) in early stages of disease point to potential defects in protein transport through the connecting cilia [START_REF] Menotti-Raymond | Mutation in CEP290 discovered for cat model of human retinal degeneration[END_REF].

In contrast to rdAC, the rd16 mouse displays an early-onset retinal dystrophy, caused by a homozygous in-frame deletion of 897 bp (AAs 1599-1897) that covers the majority of the myosin-tail homology region. Early fundus examinations revealed white retinal vessels and large pigment patches, in addition to severely reduced ERG responses for both rods and cones. The mutation causes early progressive degeneration of the OS and reduction in thickness of the outer nuclear layer [START_REF] Chang | In-frame deletion in a novel centrosomal/ciliary protein CEP290/NPHP6 perturbs its interaction with RPGR and results in early-onset retinal degeneration in the rd16 mouse[END_REF]. In addition, considerable thickening of the inner nuclear and plexiform layer were observed in mid-and central retinal regions, together with an enlargement of nuclei of all retinal cell types [START_REF] Cideciyan | Centrosomal-ciliary gene CEP290/NPHP6 mutations result in blindness with unexpected sparing of photoreceptors and visual brain: implications for therapy of Leber congenital amaurosis[END_REF] [START_REF] Acland | Gene therapy restores vision in a canine model of childhood blindness[END_REF][START_REF] Bainbridge | Effect of gene therapy on visual function in Leber's congenital amaurosis[END_REF][START_REF] Hauswirth | Treatment of leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: short-term results of a phase I trial[END_REF][START_REF] Maguire | Safety and efficacy of gene transfer for Leber's congenital amaurosis[END_REF][START_REF] Maguire | Age-dependent effects of RPE65 gene therapy for Leber's congenital amaurosis: a phase 1 dose-escalation trial[END_REF].

Clinical and diagnostic relevance unrelated LCA patients revealed its presence in 16 additional probands (21%), suggesting a major involvement of CEP290 in LCA (den [START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Under review[END_REF]. Subsequent studies corroborate that CEP290 mutations are highly recurrent in Northwestern Europe [START_REF] Perrault | Spectrum of NPHP6/CEP290 mutations in Leber congenital amaurosis and delineation of the associated phenotype[END_REF][START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Under review[END_REF], but contribute to only a minor part of LCA in Italy, Saudi Arabia, Spain, Southern India and Korea (targeted screening of c.2991+1655A>G in the latter three populations) [START_REF] Simonelli | Clinical and molecular genetics of Leber's congenital amaurosis: a multicenter study of Italian patients[END_REF][START_REF] Vallespin | Frequency of CEP290 c.2991_1655A>G mutation in 175 Spanish families affected with Leber congenital amaurosis and early-onset retinitis pigmentosa[END_REF][START_REF] Seong | Molecular characterization of Leber congenital amaurosis in Koreans[END_REF][START_REF] Li | Mutation survey of known LCA genes and loci in the Saudi Arabian population[END_REF][START_REF] Sundaresan | Mutations that are a common cause of Leber congenital amaurosis in northern America are rare in Southern India[END_REF]. Interestingly, c.2991+1655A>G was not identified in 126 Spanish patients with earlyonset RP, suggesting specificity of this mutation for LCA [START_REF] Vallespin | Frequency of CEP290 c.2991_1655A>G mutation in 175 Spanish families affected with Leber congenital amaurosis and early-onset retinitis pigmentosa[END_REF].

The CEP290-related phenotype consists of a severe cone-rod type retinal dystrophy [START_REF] Perrault | Spectrum of NPHP6/CEP290 mutations in Leber congenital amaurosis and delineation of the associated phenotype[END_REF], with best-corrected visual acuity of counting fingers or worse in the majority of cases [START_REF] Walia | Visual Acuity in Patients with Leber's Congenital Amaurosis and Early Childhood-Onset Retinitis Pigmentosa[END_REF]. Early fundus changes include white dots or a marbleized or salt and pepper aspect, and progress to midperipheral nummular or spicular pigmentation [START_REF] Littink | A novel nonsense mutation in CEP290 induces exon skipping and leads to a relatively mild retinal phenotype[END_REF][START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Under review[END_REF][START_REF] Perrault | Spectrum of NPHP6/CEP290 mutations in Leber congenital amaurosis and delineation of the associated phenotype[END_REF][START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Under review[END_REF]. A detailed study of the retinal architecture of human CEP290-mutant retinas identified profound retinal remodeling in the peripheral rod-rich regions, which was characterized by thickening of inner retinal layers. In the cone-rich foveal region, however, no clear alterations were observed. This difference in rod and cone degeneration may point to a distinct function of CEP290 in both cell types [START_REF] Cideciyan | Centrosomal-ciliary gene CEP290/NPHP6 mutations result in blindness with unexpected sparing of photoreceptors and visual brain: implications for therapy of Leber congenital amaurosis[END_REF]. Spectral-domain optical coherence tomography confirmed preservation of the outer nuclear layer in the central macula with distorted inner retina [START_REF] Pasadhika | Differential Macular Morphology in Patients with RPE65, CEP290, GUCY2D and AIPL1 Related Leber Congenital Amaurosis[END_REF]. No abnormalities were seen in visual brain pathway anatomy [START_REF] Cideciyan | Centrosomal-ciliary gene CEP290/NPHP6 mutations result in blindness with unexpected sparing of photoreceptors and visual brain: implications for therapy of Leber congenital amaurosis[END_REF]. Apart from the ocular phenotype, neurological involvement (mental retardation [MR] or autism) was observed in approximately 11-33% of cases with CEP290-related LCA, which is more frequent than in patients with mutations in other LCA genes [START_REF] Hanein | Leber congenital amaurosis: comprehensive survey of the genetic heterogeneity, refinement of the clinical definition, and genotype-phenotype correlations as a strategy for molecular diagnosis[END_REF][START_REF] Perrault | Spectrum of NPHP6/CEP290 mutations in Leber congenital amaurosis and delineation of the associated phenotype[END_REF] Coppieters , 2010). Four probands presented with either (transitory) hypotonia or ataxia [START_REF] Perrault | Spectrum of NPHP6/CEP290 mutations in Leber congenital amaurosis and delineation of the associated phenotype[END_REF]. Interestingly, some patients displayed signs suggestive for involvement of other ciliary processes. One patient presented with recurrent otitis media [START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Under review[END_REF], and LCA patients homozygous for c.2991+1655A>G exhibited severe olfactory dysfunction, whereas mild to severe microsmia was observed in heterozygous carriers [START_REF] Mcewen | Hypomorphic CEP290/NPHP6 mutations result in anosmia caused by the selective loss of G proteins in cilia of olfactory sensory neurons[END_REF]. Obviously, MR, autism, ataxia and hypotonia may be signs of the mild end of the clinical spectrum of systemic phenotypes related to CEP290 rather than of true LCA as isolated retinal disease.

Nephronophthisis (NPHP) and Senior-Loken syndrome (SLS)

Nephronophthisis (NPHP, MIM[256100]) is the most frequent genetic cause for end-stage renal disease (ESRD) in the first three decades of life. Infantile, juvenile and adolescent forms have been described. Key histology findings are tubulointerstitial fibrosis, tubular atrophy, tubular dilatation and cyst formation. Renal ultrasound (US) reveals increased cortical echogenicity, loss of corticomedullary differentiation and corticomedullary cysts [START_REF] Salomon | Nephronophthisis[END_REF][START_REF] Simms | Nephronophthisis[END_REF]. In 10-15% of cases, juvenile NPHP is accompanied by retinal involvement, which is called Senior-Loken syndrome (SLS, MIM [266900]). Based on the degree of retinal degeneration, both early-onset and late-onset types have been described. NPHP and SLS are AR disorders. So far, nine genes are known to cause NPHP; some of them are associated with SLS [START_REF] Hildebrandt | Nephronophthisis-associated ciliopathies[END_REF][START_REF] Hildebrandt | Nephronophthisis: disease mechanisms of a ciliopathy[END_REF]. CEP290 mutations have been described

in eight families with SLS, two of which present with intrafamilial clinical variability of neurological and/or renal involvement [START_REF] Tory | High NPHP1 and NPHP6 mutation rate in patients with Joubert syndrome and nephronophthisis: potential epistatic effect of NPHP6 and AHI1 mutations in patients with NPHP1 mutations[END_REF]. In six families, two mutations segregated, while in two other families, one heterozygous CEP290 mutation occurred in combination with a homozygous mutation in NPHP1 and a heterozygous mutation in NPHP4, respectively [START_REF] Helou | Mutation analysis of NPHP6/CEP290 in patients with Joubert syndrome and Senior-Loken syndrome[END_REF][START_REF] Tory | High NPHP1 and NPHP6 mutation rate in patients with Joubert syndrome and nephronophthisis: potential epistatic effect of NPHP6 and AHI1 mutations in patients with NPHP1 mutations[END_REF]. The age at which ESRD occurred ranged from 5 to 40 years and retinal degeneration was usually severe [START_REF] Sayer | The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4[END_REF][START_REF] Helou | Mutation analysis of NPHP6/CEP290 in patients with Joubert syndrome and Senior-Loken syndrome[END_REF][START_REF] Tory | High NPHP1 and NPHP6 mutation rate in patients with Joubert syndrome and nephronophthisis: potential epistatic effect of NPHP6 and AHI1 mutations in patients with NPHP1 mutations[END_REF][START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Under review[END_REF]. It is presumed that CEP290 mutations do not represent a major cause of NPHP/SLS, since large-scale mutation screening in almost 100 families with SLS [START_REF] Helou | Mutation analysis of NPHP6/CEP290 in patients with Joubert syndrome and Senior-Loken syndrome[END_REF][START_REF] O'toole | Mutational analysis in 119 families with nephronophthisis[END_REF] and 21 families with isolated NPHP revealed mutations in only two patients with SLS and a heterozygous unclassified variant in one patient with NPHP [START_REF] Helou | Mutation analysis of NPHP6/CEP290 in patients with Joubert syndrome and Senior-Loken syndrome[END_REF].

Joubert syndrome (JS) and Joubert syndrome related disorders (JSRD)

Joubert syndrome (JS, MIM [213300]) is a genetically heterogeneous disorder of which the main phenotypic hallmarks are hypotonia, ataxia, psychomotor delay and variable occurrence of oculomotor apraxia and neonatal breathing abnormalities. The most consistent feature, the "molar tooth sign" (MTS), is visible on magnetic resonance imaging (MRI) and consists of a deep interpeduncular fossa with narrow isthmus, thickened, elongated and mal-oriented superior cerebellar peduncles and cerebellar vermis aplasia/hypoplasia [START_REF] Doherty | Joubert syndrome: insights into brain development, cilium biology, and complex disease[END_REF]. In addition to pure JS, a range of disorders exists that share the MTS and the main neurological signs of JS, yet are distinct due to the involvement of other organs. They are called Joubert syndrome-related disorders (JSRD) and consist of five major subgroups: JS associated with retinopathy, JS associated with renal involvement, cerebello-oculo-renal syndrome (CORS or JS-SLS), COACH syndrome (MIM [216360]) and orofaciodigital syndrome type VI (OFDVI, MIM[277170]) [START_REF] Valente | Genotypes and phenotypes of Joubert syndrome and related disorders[END_REF].

Currently, less than 50% of AR cases can be attributed to mutations in nine genes, which all encode ciliary proteins, as far as known [START_REF] Valente | Genotypes and phenotypes of Joubert syndrome and related disorders[END_REF][START_REF] Bielas | Mutations in INPP5E, encoding inositol polyphosphate-5-phosphatase E, link phosphatidyl inositol signaling to the ciliopathies[END_REF][START_REF] Doherty | Joubert syndrome: insights into brain development, cilium biology, and complex disease[END_REF][START_REF] Valente | Mutations in TMEM216 perturb ciliogenesis and cause Joubert, Meckel and related syndromes[END_REF]. CEP290 mutations are a major cause of JSRD, in particular the CORS [START_REF] Valente | Mutations in CEP290, which encodes a centrosomal protein, cause pleiotropic forms of Joubert syndrome[END_REF][START_REF] Brancati | CEP290 mutations are frequently identified in the oculo-renal form of Joubert syndrome-related disorders[END_REF][START_REF] Helou | Mutation analysis of NPHP6/CEP290 in patients with Joubert syndrome and Senior-Loken syndrome[END_REF][START_REF] Tory | High NPHP1 and NPHP6 mutation rate in patients with Joubert syndrome and nephronophthisis: potential epistatic effect of NPHP6 and AHI1 mutations in patients with NPHP1 mutations[END_REF][START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Under review[END_REF].

Meckel-Grüber syndrome (MKS)

Meckel-Grüber syndrome (MKS, MIM[249000]) is a neonatally lethal disorder characterized by a combination of central nervous system malformations (typically occipital

[meningo]encephalocele), bilateral cystic kidney dysplasia, ductal proliferation in the portal area of the liver and postaxial polydactyly. Associated features may include cardiac abnormalities, developmental anomalies of external or internal genitalia, cleft palate, situs inversus and others.

The worldwide incidence ranges from 1/13,250 to 1/140,000 live births [START_REF] Alexiev | Meckel-Gruber syndrome: pathologic manifestations, minimal diagnostic criteria, and differential diagnosis[END_REF].

Some cases present with incomplete phenotypes that are called "Meckel-like syndrome"

(MIM [208540]) and probably make up a clinical spectrum situated between JSRD and MKS. In addition, a phenotypic and genetic overlap was seen between MKS and BBS (see "Bardet-Biedl Deleted: patients Deleted: one Deleted: However, renal failure at an age of 4 and 25 was also reported [START_REF] Brancati | CEP290 mutations are frequently identified in the oculo-renal form of Joubert syndrome-related disorders[END_REF][START_REF] Tory | High NPHP1 and NPHP6 mutation rate in patients with Joubert syndrome and nephronophthisis: potential epistatic effect of NPHP6 and AHI1 mutations in patients with NPHP1 mutations[END_REF].

Retinal degeneration mostly consisted of congenital blindness, although RP has also been described in a few patients [START_REF] Brancati | CEP290 mutations are frequently identified in the oculo-renal form of Joubert syndrome-related disorders[END_REF][START_REF] Helou | Mutation analysis of NPHP6/CEP290 in patients with Joubert syndrome and Senior-Loken syndrome[END_REF].

Deleted: . These features include et al., 2005;[START_REF] Leitch | Hypomorphic mutations in syndromic encephalocele genes are associated with Bardet-Biedl syndrome[END_REF]. MKS is inherited in an AR fashion with five genes (six loci) identified so far, all encoding centrosomal/ciliary proteins.

Mutations in CEP290 were identified in eight families with MKS and four families with Meckellike syndrome, further supporting a role for CEP290 in a wide range of phenotypes (Baala et al., 2007a;[START_REF] Frank | Mutations of the CEP290 gene encoding a centrosomal protein cause Meckel-Gruber syndrome[END_REF].

Bardet-Biedl syndrome (BBS)

Bardet-Biedl syndrome (BBS, MIM[209900] is a complex multi-organ ciliopathy, characterized by a variable combination of retinal degeneration, obesity, hypogonadism, polydactyly, renal dysfunction and MR. Additional features consist of neurological impairment, speech deficits, craniofacial abnormalities, hearing loss, diabetes mellitus, metabolic defects, cardiovascular abnormalities, hepatic defects and Hirschsprung disease. In most cases, BBS is inherited as an AR trait, with currently 14 genes known. Notably, mutations in different genes have been described in the same patient. These modifiers affect either the expressivity or the overall penetrance of the phenotype (Zaghloul and Katsanis, 2009). In search of a genetic explanation for the partial clinical overlap between BBS and MKS, Leitch and coworkers identified MKS1 and CEP290 as disease genes for BBS. Interestingly, the homozygous p.Glu1903X CEP290 mutation causing BBS was found together with a complex TMEM67 allele, possibly influencing the phenotype [START_REF] Leitch | Hypomorphic mutations in syndromic encephalocele genes are associated with Bardet-Biedl syndrome[END_REF].

Genotype-phenotype correlations

For the majority of mutations, no clear-cut correlation could be established between the genotype and clinical expression. Despite 90 mutations reported exclusively in only one phenotype, 14 others segregated with two diseases while 8 were even associated with three or more phenotypes. In most cases, these phenotypes are partially overlapping, although few mutations were observed to lead to strongly divergent disorders, such as LCA and MKS. Overall, mutations causing JS(RD) tend to cluster in the second half of the gene, whereas mutations segregating with LCA, SLS and MKS are homogeneously distributed throughout the gene [START_REF] Brancati | CEP290 mutations are frequently identified in the oculo-renal form of Joubert syndrome-related disorders[END_REF][START_REF] Valente | Genotypes and phenotypes of Joubert syndrome and related disorders[END_REF] (Supp. Table S1).

For a few mutations, a genotype-phenotype correlation could be established, albeit to a limited extent. The most common mutation in JSRD, p.Gly1890X, was also described in two siblings with isolated LCA, in compound heterozygosity with c.2991+1655A>G [START_REF] Cideciyan | Centrosomal-ciliary gene CEP290/NPHP6 mutations result in blindness with unexpected sparing of photoreceptors and visual brain: implications for therapy of Leber congenital amaurosis[END_REF].

Despite the severe retinal phenotype of these siblings and four other patients heterozygous for Gly1890X, a milder or even absent visual impairment was seen in eight of nine patients homozygous for p.Gly1890X, suggesting that this mutation might be less harmful for retinal function [START_REF] Sayer | The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4[END_REF][START_REF] Valente | Mutations in CEP290, which encodes a centrosomal protein, cause pleiotropic forms of Joubert syndrome[END_REF][START_REF] Brancati | CEP290 mutations are frequently identified in the oculo-renal form of Joubert syndrome-related disorders[END_REF][START_REF] Valente | Genotypes and phenotypes of Joubert syndrome and related disorders[END_REF].

Similarly, a compound heterozygous genotype c.2991+1655G>A/c.451C>T caused an earlyonset retinal dystrophy phenotype less severe than most CEP290-related LCA, probably due to the mild effect of both mutations [START_REF] Littink | A novel nonsense mutation in CEP290 induces exon skipping and leads to a relatively mild retinal phenotype[END_REF]. So far, c.2991+1655A>G has only been described in patients with isolated LCA. Since a small amount of wild-type product is still present, a dosage-dependent mechanism was proposed, in which complete loss of function of both alleles would lead to JS, whereas a residual CEP290 activity would be sufficient for normal cerebellar and renal function but not for correct retinal activity (den [START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Under review[END_REF].

However, subsequent identification of truncating mutations on both alleles in several LCA patients countered the latter hypothesis [START_REF] Perrault | Spectrum of NPHP6/CEP290 mutations in Leber congenital amaurosis and delineation of the associated phenotype[END_REF]. In addition, we identified c.2991+1655A>G in two patients with features suggestive of renal dysfunction, implicating that this mutation is possibly not exclusively associated with isolated LCA [START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Under review[END_REF]. [START_REF] Tory | High NPHP1 and NPHP6 mutation rate in patients with Joubert syndrome and nephronophthisis: potential epistatic effect of NPHP6 and AHI1 mutations in patients with NPHP1 mutations[END_REF]. In addition, a homozygous splice site mutation segregated with SLS in a Turkish family [START_REF] Sayer | The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4[END_REF]. However, in two other unrelated patients with neurological involvement, two distinct splice site mutations were detected. Of note, in both cases, one of both mutations does not affect a consensus splice site (c.1711+5A>G and c.6271-8T>G) [START_REF] Perrault | Spectrum of NPHP6/CEP290 mutations in Leber congenital amaurosis and delineation of the associated phenotype[END_REF][START_REF] Tory | High NPHP1 and NPHP6 mutation rate in patients with Joubert syndrome and nephronophthisis: potential epistatic effect of NPHP6 and AHI1 mutations in patients with NPHP1 mutations[END_REF].

For all CEP290-related phenotypes, different degrees of neurological, ocular and renal involvement were observed between unrelated patients harboring the same CEP290 genotype. A homozygous p.Lys1575X mutation was detected in four patients with isolated LCA and normal development, one patient with LCA and autistic behavior but normal MRI, one patient with LCA and severe MR (of whom no MRI was available), and one patient with LCA-JS (proven MTS on MRI) [START_REF] Perrault | Spectrum of NPHP6/CEP290 mutations in Leber congenital amaurosis and delineation of the associated phenotype[END_REF][START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Under review[END_REF]. Patients homozygous for p.Gly1890X always displayed characteristics typical of JS but associated features ranged from none over JS with NPHP to CORS [START_REF] Sayer | The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4[END_REF][START_REF] Valente | Mutations in CEP290, which encodes a centrosomal protein, cause pleiotropic forms of Joubert syndrome[END_REF][START_REF] Brancati | CEP290 mutations are frequently identified in the oculo-renal form of Joubert syndrome-related disorders[END_REF]. A homozygous p.Trp7Cys mutation was identified in two patients of Pakistani origin who suffered from retinal degeneration and NPHP. In only one patient, however, several features suggestive for JS were observed [START_REF] Valente | Mutations in CEP290, which encodes a centrosomal protein, cause pleiotropic forms of Joubert syndrome[END_REF][START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Under review[END_REF]. In addition, compound French siblings with CORS [START_REF] Tory | High NPHP1 and NPHP6 mutation rate in patients with Joubert syndrome and nephronophthisis: potential epistatic effect of NPHP6 and AHI1 mutations in patients with NPHP1 mutations[END_REF], and two French siblings with Meckel-like syndrome, aged 18 and 29 weeks of gestation, respectively (Baala et al., 2007a). Moreover, intrafamilial variability was reported in several cases. Perrault and colleagues described two LCA families in which all affected sibs carried the same mutations, but displayed different neurological involvement [START_REF] Perrault | Spectrum of NPHP6/CEP290 mutations in Leber congenital amaurosis and delineation of the associated phenotype[END_REF]. In addition, kidney US in two brothers with JS (deceased at an age of four and seven months), both homozygous for p.Gln1942X, revealed cortical cysts in only one patient [START_REF] Valente | Mutations in CEP290, which encodes a centrosomal protein, cause pleiotropic forms of Joubert syndrome[END_REF]. Overall, this wide clinical spectrum is difficult to explain by the CEP290 genotype alone.

Epistatic effect of other components of the ciliary proteome

It is presumed that the clinical variability of CEP290-related disease might be caused by secondsite modifier alleles. Since CEP290 forms a complex with several members of the ciliary proteome, variants in these genes are likely to affect the interaction with and function of CEP290.

So far, three ciliary genes have been described in which variants co-occur with CEP290 mutations. The first gene is AHI1, which is associated with JS(RD) and possibly acts in a pathway common with CEP290 [START_REF] Ferland | Abnormal cerebellar development and axonal decussation due to mutations in AHI1 in Joubert syndrome[END_REF][START_REF] Parisi | AHI1 mutations cause both retinal dystrophy and renal cystic disease in Joubert syndrome[END_REF][START_REF] Kim | CEP290 interacts with the centriolar satellite component PCM-1 and is required for Rab8 localization to the primary cilium[END_REF][START_REF] Hsiao | Ahi1, whose human ortholog is mutated in Joubert syndrome, is required for Rab8a localization, ciliogenesis and vesicle trafficking[END_REF]. Tory and colleagues identified a heterozygous p.Arg830Trp missense variant in a CORS patient harboring a homozygous p.Leu1884ThrfsX23 CEP290 mutation. This AHI1 variant was not present in an affected sibling with the same CEP290 genotype. Interestingly, no significant difference could be seen in neurological and ocular manifestation between both sibs, albeit that the one carrying p.Arg830Trp displayed renal failure earlier than the other (11 versus 25 years). In addition, a higher frequency of p.Arg830Trp was observed in patients with NPHP1 mutations and neurological symptoms, in comparison with patients with NPHP1 mutations with JS-related neurological involvement in up to 12% of cases [START_REF] Tory | High NPHP1 and NPHP6 mutation rate in patients with Joubert syndrome and nephronophthisis: potential epistatic effect of NPHP6 and AHI1 mutations in patients with NPHP1 mutations[END_REF]. Recently, a modifying effect of p.Arg830Trp has also been established on the development of retinal degeneration in patients suffering from NPHP, independent of primary mutations in NPHP1.

AHI1 genetically interacts with NPHP1 in retinal development and was proven necessary for photoreceptor OS development [START_REF] Louie | AHI1 is required for photoreceptor outer segment development and is a modifier for retinal degeneration in nephronophthisis[END_REF]. In addition to p.Arg830Trp, we identified a different heterozygous missense variant, p.Asn811Lys, in the most severely affected patient out of three with the same CEP290 genotype and LCA but with different neurological involvement as well as variable age-of-onset of ESRD. Yet another LCA patient with MR carried a third AHI1 missense variant, p.His758Pro, in combination with two mutations in CEP290 [START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Under review[END_REF]. Of note, all potential modifier alleles identified in AHI1 so far have been missense

variants, located in the strongly conserved WD40 repeat, whereas most of the JS-causing mutations are truncating. Obviously, these variants in AHI1 are not sufficient to explain all of the clinical variability, as no AHI1 mutations were demonstrated in two large cohorts of patients with JS(RD) and CEP290 mutations [START_REF] Brancati | CEP290 mutations are frequently identified in the oculo-renal form of Joubert syndrome-related disorders[END_REF][START_REF] Helou | Mutation analysis of NPHP6/CEP290 in patients with Joubert syndrome and Senior-Loken syndrome[END_REF]. A second gene considered to harbor a potential modifier allele is TMEM67 (MKS3). A homozygous p.Glu1903X CEP290 mutation was accompanied by a complex p.[Gly218Ala; Ser320Cys] TMEM67 allele in a patient with BBS. The TMEM67 allele is likely to influence the CEP290-related phenotype,

given the genetic interaction between cep290 and Tmem67 in zebrafish and the pathogenic potential of the p.Ser320Cys variant and to a lesser extent the p.Gly218Ala variant [START_REF] Leitch | Hypomorphic mutations in syndromic encephalocele genes are associated with Bardet-Biedl syndrome[END_REF]. NPHP4, which is associated with both isolated NPHP and SLS [START_REF] Otto | A gene mutated in nephronophthisis and retinitis pigmentosa encodes a novel protein, nephroretinin, conserved in evolution[END_REF], is the third gene, since a heterozygous p.Thr627Met mutation in NPHP4 was identified in a patient with SLS who is also heterozygous for p.Arg1978X in CEP290 [START_REF] Hoefele | Mutational analysis of the NPHP4 gene in 250 patients with nephronophthisis[END_REF]Helou et , 2007). In addition to the heterozygous alleles identified in AHI1, TMEM67 and NPHP4, both homozygosity at the PKHD1 locus and a homozygous CEP290 mutation were identified in a consanguineous family segregating MKS as well as AR polycystic kidney disease. As expected, a homozygous CEP290 mutation combined with linkage to the PKHD1 locus in one sibling with MKS caused a more severe kidney and liver phenotype, in comparison with the other siblings that carried either the same CEP290 or PKHD1 genotype (Baala et al., 2007a).

Conversely, mutations in CEP290 might affect phenotypes caused by mutations in other ciliary genes. A heterozygous p.Asn96MetfsX29 mutation segregated in two siblings who also harbored a homozygous NPHP1 deletion. In spite of a common genotype and similar retinal degeneration, these siblings differ in both renal and neurological phenotype, suggesting the involvement of additional factors [START_REF] Tory | High NPHP1 and NPHP6 mutation rate in patients with Joubert syndrome and nephronophthisis: potential epistatic effect of NPHP6 and AHI1 mutations in patients with NPHP1 mutations[END_REF]. In addition, a heterozygous CEP290 mutation/variant was identified in two probands that might carry a homozygous mutation in another ciliary gene, since they each originate from consanguineous parents [START_REF] Helou | Mutation analysis of NPHP6/CEP290 in patients with Joubert syndrome and Senior-Loken syndrome[END_REF].

Molecular diagnostic strategies

Given the large number of coding exons (53), sequencing of CEP290 is laborious, expensive and requires a considerable amount of DNA. Therefore, first-step analysis might comprise screening for the most recurrent mutations, the choice of which depends on the phenotype of the patient.

This approach has been applied successfully by several groups (den [START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Under review[END_REF][START_REF] Perrault | Spectrum of NPHP6/CEP290 mutations in Leber congenital amaurosis and delineation of the associated phenotype[END_REF][START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Under review[END_REF]. Targeted mutation screening can be performed testing for 641 mutations in 13 genes (Asper Ophthalmics, v8.0). In the majority of cases however, screening of the complete coding region remains necessary, either to identify the second disease allele, or to screen for both mutations in case of negative first-pass screening.

PCR primers and conditions for both sequencing [START_REF] Sayer | The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4[END_REF][START_REF] Valente | Mutations in CEP290, which encodes a centrosomal protein, cause pleiotropic forms of Joubert syndrome[END_REF] and dHPLC [START_REF] Brancati | CEP290 mutations are frequently identified in the oculo-renal form of Joubert syndrome-related disorders[END_REF] have been published and an alternative pre-screening assay based on heteroduplex formation and subsequent CEL I endonuclease digest has been described [START_REF] Helou | Mutation analysis of NPHP6/CEP290 in patients with Joubert syndrome and Senior-Loken syndrome[END_REF]. Brancati and colleagues first screened parental DNA using dHPLC, followed by sequencing of the identified mutations in the affected offspring and of the whole coding region in case of a heterozygous mutation [START_REF] Brancati | CEP290 mutations are frequently identified in the oculo-renal form of Joubert syndrome-related disorders[END_REF]. Apart from detecting small variants, comprehensive CEP290 screening should also include dosage analysis of all exons, using for instance quantitative real-time PCR (Travaglini et al., 2009).

Future Prospects

Given the emerging importance of modifier alleles on the phenotypic expression of ciliopathies, future studies are required to understand their mechanism of action and to further elucidate the ciliary protein networks. The identification of modifiers for retinal, renal, neurological or other phenotypes might contribute to an improved predictive power of a CEP290-related genotype.

Early management of patients with a higher risk of NPHP, for instance, may delay the progression toward renal failure and minimize secondary complications. In a diagnostic setting, mutations in ciliopathies should ideally be evaluated in the context of the entire spectrum of ciliary variants. Novel technologies such as next-generation sequencing will play a crucial role in this respect. Finally, more insights in the molecular pathogenesis of ciliopathies will eventually direct towards therapeutic options, such as gene therapy. The exact location of the mutation/variant is depicted with respect to the CEP290 protein [START_REF] Sayer | The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4[END_REF]. If no protein effect is known, the c.DNA nomenclature is used. Mutations depicted twice have been reported to arise from different nucleotide changes. Supp. Table S2: Overview of all CEP290 unclassified variants identified so far and their occurrence in different phenotypes. Supp. Figure S1: Overview of CEP290base. The database is accessible from: http://medgen.ugent.be/cep290base. Information on variants can be retrieved using the Mutation Overview (1) or the Mutation Browser (2). In addition, the Phenotype page (3) describes all phenotypes associated with CEP290 so far. The variant-specific pages (4) are accessible from both the Mutation Overview and Mutation Browser and include detailed information on the pathogenic potential. The database links to NetGene2 and BDGP for 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, thereby providing a unique opportunity to link modifiers to associated clinical manifestations.

Page 51 of 51

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Human Mutation

  and p.Phe1950LeufsX15 mutations were described in two

  In the first family, only one of two affected sibs suffered from autism, whereas in the other family, two out of five affected sibs were mentally retarded Deleted: or with healthy controls, suggesting an influence on the neurological expression of NPHP1-related disease. Mutations in NPHP1 cause NPHP or SLS,

Figure 1 :

 1 Figure 1: Overview of all mutations and variants currently present in CEP290base.

Figure 2 :Figure 1 :Figure 1 :Figure 2 :Figure 2 :

 21122 Figure 2: Overlap of CEP290 mutations between different diseases. Abbreviations used: LCA: Leber Congenital Amaurosis; SLS: Senior-Loken syndrome; JS: Joubert syndrome; JS + RD: Joubert syndrome with associated retinopathy; JS + RF: Joubert syndrome with associated renal failure; CORS: cerebello-oculo-renal syndrome; MKS: Meckel-Grüber syndrome; MKS-like: Meckel-Grüber syndrome -like; BBS: Bardet-Biedl syndrome.

  Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence (NM_025114.3), according to journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1.Page 7 number of families. Abbreviations used: LCA: Leber Congenital Amaurosis; SLS: Senior-Loken syndrome; JS: Joubert syndrome; JS + RD: Joubert syndrome with associated retinopathy; JS + RF: Joubert syndrome with associated renal failure; CORS: cerebello-oculo-renal syndrome;MKS: Meckel-Grüber syndrome; MKS-like: Meckel-Grüber syndrome-like; BBS: Bardet-Biedl syndrome. ¥: nucleotide nomenclature not specified in publication. *: both patients carrying the mutation belong to the same family.Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence (NM_025114.3), according to journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1. Page 8

Table 2

 2 Deleted: F

	Deleted: four

9 al., 2010). Two additional unclassified variants include in-frame deletions. A p.Glu1554del variant segregated with p.Phe1950LeufsX15 in an LCA patient

Table 2

 2 Deleted: the locus-specific mutation database

	Deleted: is
	Deleted: o

  . Interestingly, rd16 mice displayed severe early-onset olfactory dysfunction. Although the olfactory sensory neurons have with correct localization of CEP290 to dendritic knobs, two components of the olfactory G protein that are in complex with CEP290, G γ13 and G olf , were undetectable in the cilia of rd16 mice, suggesting a potential role for CEP290 in the regulation of olfactory G protein trafficking[START_REF] Mcewen | Hypomorphic CEP290/NPHP6 mutations result in anosmia caused by the selective loss of G proteins in cilia of olfactory sensory neurons[END_REF].Both models offer interesting opportunities for in vivo research on CEP290 function and therapeutic intervention. The progressive nature of retinal disease in rd16 mice resembles human LCA, and therefore constitutes an excellent basis to study disease development. Moreover, the existence of a large animal model such as rdAC is an asset for the implementation of genespecific therapy for retinal degeneration, as already shown by successful RPE65-replacement therapy in Briard dogs which preceded human phase I clinical trials

	structurally intact cilia
		Deleted: Ensembl
		Deleted: causal gene
		Deleted:
		Deleted: the inclusion of an additional
		4 bp
		Deleted: -insertion
		Deleted: at the end of
	F o	Deleted: m
	r	Deleted: The age of onset of the first
	P	ophthalmoscopic signs is highly variable, with 12-18 months of age in the majority of animals
	e e r
	R e v i e w	Deleted: a Deleted: functions
		Deleted: displayed
	17

  CEP290 was identified as a disease gene for LCA, with the deep intronic substitution c.2991+1655A>G as causal mutation. Targeted screening of this mutation in 76

	Page 17 of 51	Human Mutation
			Deleted: ACIII, CNGA2, and different
			G protein-coupled olfactory receptors
			Deleted: . These data suggest
			Deleted: into cilia
	F o		Deleted: preceding
	r
	P
	e e r
		R e v i e w	Deleted: In general, LCA is inherited in an AR manner. Deleted: approximately Deleted: causal
			Deleted: the initial
		18
	John Wiley & Sons, Inc.

CEP290-linked phenotypes

Leber Congenital Amaurosis (LCA)

Leber Congenital Amaurosis (LCA, MIM

[204000]

) is the earliest and most severe form of all inherited retinal dystrophies, causing profound visual deficiency, nystagmus and an undetectable or severely reduced ERG in the first year of life. Approximately 20% of all blind children are thought to suffer from this disease, which is mainly inherited in an AR manner. So far, 15 disease loci (14 genes) are known, which together account for ~70% of cases (den

[START_REF] Hollander | Leber congenital amaurosis: genes, proteins and disease mechanisms[END_REF]

. In 2006,

  was proposed for certain other CEP290 splice site mutations. It was presumed that these do not lead to truncated proteins, but rather to (partial) deletion of one of the CCs. In contrast to truncating mutations, these do not seem to hamper normal neurologic development. Examples are two first-degree cousins, each carrying p.Leu1884ThrfsX23 on one allele, but a different CEP290 mutation on the other. One of them harbored a nonsense mutation and presented with JS without renal involvement by the age of eight, whereas the other was heterozygous for c.4195-1G>A and suffered from SLS without neurologic symptoms

	A similar hypothesis
		Deleted: in
		Deleted: M
		Deleted: Table 1
		Deleted: Interestingly, t
	F o
	r
	P
	e e r	Deleted: the Deleted: mutation
	R e v i e w	Deleted: for
	24

  Since all CEP290-related phenotypes

		Deleted: LCA
	are inherited in an AR manner and the majority of mutations consist of loss-of-function alleles, Zaghloul NA, Katsanis N. 2009. Mechanistic insights into Bardet-Biedl syndrome, a model
	ciliopathy. J Clin Invest 119(3):428-37.	Deleted: early-onset RP and LCA
	gene-replacement therapy might indeed be an option.
		Deleted: purposes
		Deleted: In order to preserve DNA of
		affected children,
	F o F o F o F o F o F o F o F o
	r r r r r r r r
	P P P P P P P P
	e e r e e r e e r e e r e e r e e r e e r e e r	Deleted: necessary
	R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w	Deleted: clinical
	29 36

Table 1 :

 1 Overview of selected ciliary proteins interacting with CEP290, and their involvement in partially overlapping yet distinct ciliopathies.The database is accessible from: http://medgen.ugent.be/cep290base. Information on variants can be retrieved using the Mutation Overview (1) or the Mutation Browser (2). In addition, the Phenotype page (3) describes all phenotypes associated with CEP290 so far. The variant-specific pages (4) are accessible from both the Mutation Overview and Mutation Browser and include detailed information on the pathogenic potential. The database links to NetGene2 and BDGP for 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, thereby providing a unique opportunity to link modifiers to associated clinical manifestations.

	Page 41 of 51
									Deleted: 3
		Protein	RD	NPHP	Associated phenotype SLS JS(RD) MKS	BBS	COACH
						(Sayer,
		CEP290	(den Hollander et al., 2006)	(Helou et al., 2007)(?)	(Sayer et al., 2006)	et al., 2006; Valente et al.,	(Baala et al., 2007a)	(Leitch et al., 2008)
						2006)
		RPGR	(Meindl et al., 1996)
	CEP290 interacating proteins	RPGRIP1 NPHP5 TMEM67 (MKS3)	(Dryja et al., 2001)		(Otto et al., 2005)	(Baala et al., 2007b) (Gorden	(Smith et al., 2006) (Tallila		(Brancati et al., 2009) (Doherty	Deleted: MKS3 ¶ Deleted: TMEM67
		CC2D2A				et al.,	et al.,		et al.,
						2008)	2008)		2009)

Abbreviations used: RD: retinal degeneration; NPHP: nephronophthisis; SLS: Senior-Loken syndrome; JS(RD): Joubert syndrome (related disorders); MKS: Meckel-Grüber syndrome; BBS: Bardet-Biedl syndrome; COACH: cerebellar vermis hypo/aplasia, oligophrenia, congenital ataxia, ocular coloboma, and hepatic fibrosis; (?): uncertain association.

Table S1 : Overview of all CEP290 mutations identified so far and their occurrence in different phenotypes.

 S1

	Mutation				Associated phenotype
	Nucleotide nomenclature	Protein nomenclature	LCA	SLS	JS	JS + RD	JS + RF	CORS MKS	MKS-like	BBS
	c.1A>G	p.Met1?		1
	c.2T>A	p.Met1?	1
	c.21G>T	p.Trp7Cys		1				1
	c.103-13_103-18del	p.?						1
	c.164_167del	p.Thr55SerfsX3						1
	c.180+1G>T	p.?	1
	c.180+2T>A	p.?						2 (1)
	c.265dup	p.Thr89AsnfsX2	1
	c.287del	p.Asn96MetfsX29		1*		1*
	c.322C>T	p.Arg108X	1
	c.381_382delinsT	p.Lys127AsnfsX36						1
	c.384_385del	p.Asp128GlufsX17	1
	c.384_387del	p.Asp128GlufsX34	1					4 (2)
	c.437del	p.Glu146GlyfsX17	1
	c.451C>T	p.Arg151X	1
	c.566C>G	p.Ser189X	1
	c.613C>T	p.Arg205X						2 (1)
	c.679_680del	p.Glu227SerfsX2	1
	c.1066-1G>A	p.?						1
	c.1189+1G>A	p.?	1
	c.1219_1220del	p.Met407GlufsX14	3					1
	c.1260_1264del	p.Lys421GlyfsX2	2
	c.1361del	p.Gly454GlufsX5						1
	c.1419_1423del	p.Ile474ArgfsX5		1
	c.1550del	p.Leu517X	1
	c.1593C>A	p.Tyr531X	1
	c.1645C>T	p.Arg549X				1
	c.1666del	p.Ile556PhefsX17				1

Deleted: Table

1 Page 44 of 51 John Wiley & Sons, Inc.

  Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence (NM_025114.3), according to journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1.Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence (NM_025114.3), according to journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1.Page 6This table gives an overview of the number of patients carrying a specific mutation, for each phenotype (updating date: 14 th of June, 2010). Numbers between brackets indicate the unique

	Page 45 of 51			Human Mutation Human Mutation
	Mutation Mutation				Associated phenotype Associated phenotype
	Nucleotide nomenclature Nucleotide nomenclature	Protein nomenclature Protein nomenclature	LCA LCA	SLS SLS	JS JS	JS + RD JS + RD	JS + RF JS + RF	CORS MKS CORS MKS	MKS-like MKS-like	BBS BBS
	c.1682_1683del	p.Gln561ArgfsX14				1	(2)
	c.1709C>G ¥	p.Ser570X p.Glu1903X	1								1
	c.1711+5A>G CEP290:c.5709+2352_	p.?				1
	c.1824G>A 54_ C12orf50:c.290-	p.= p.?	1					1
	c.1859_1862del 1375_77del	p.Arg621IlefsX2	2
	c.1860_1861del c.5722G>T	p.Asp622PhefsX5 p.Glu1908X				1		1
	c.1910-2A>C c.5734del	p.? p.Trp1912GlyfsX11	1					4 (2)
	c.1936C>T c.5776C>T	p.Gln646X p.Arg1926X	2 (1) 1
	c.1984C>T c.5813_5817del	p.Gln662X p.Thr1938AsnfsX16	1						2 (1)
	c.1992del c.5824C>T	p.Pro665LeufsX10 p.Gln1942X	1		1*		1*
	c.2118_2122dup c.2218-15_2220del c.2218-4_2222del c.2218-2A>C c.2249T>G c.5850del c.5865_5867delinsGG c.5866G>T c.5932C>T c.5941G>T	F o r p.Thr709SerfsX9 1 p.? 1 p.? p.? 1 p.Leu750X 1 F o r p.Phe1950LeufsX15 3 F p.Glu1956GlyfsX9 1 p.Glu1956X 1 o p.Arg1978X r p.Glu1981X	2 (1) 1				1 2 (1) 2	2 (1)	Deleted: c.2118_2122dup	... [1]
	c.2251C>T c.2695C>T c.6031C>T c.6072C>A	p.Arg751X p.Gln899X p.Arg2011X p.Tyr2024X	P 1 P P		1		1 2 (1)
	c.2906dup c.2991+1655A>G c.3043G>T c.3104-2A>G c.6271-8T>G c.6277del c.6331C>T c.6604del c.3104-1G>A c.3175del c.3175dup c.3176del c.3292G>T c.3310-1G>C c.3310-1_3310delinsAA c.3422dup c.3793C>T c.6869del c.6870del c.7318_7321dup c.7341dup c.7366_7369del Total number of distinct patients (families) p.Tyr969X p.Cys998X p.Glu1015X p.? p.? p.Val2093SerfsX4 p.Gln2111X p.Ile2202LeufsX24 p.? p.Ile1059X p.Ile1059AsnfsX11 p.Ile1059LysfsX6 p.Glu1098X p.? p.? p.Leu1141PhefsX5 p.Gln1265X p.Asn2290IlefsX11 p.Gln2291LysfsX10 p.Leu2441SerfsX16 p.Leu2448ThrfsX8 p.Thr2457AlafsX27	e e r R e v i e w (83) 92 e e r 1 e 1 1 e 7 (2) r 1 1 1 1 1 1 2 1 3 1 1 R e v i e w 2 2 (1) R 2 (1) 1 2 e 2 (1) v i e w 184 (167) 13 (12) 2 23 (20 ) 4 (3) 65 (53) 17 (11)	1 1 1 1 10 (7)	1
	Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG
	translation initiation codon in the reference sequence (NM_025114.3), according to journal
	guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1.			Page 4
			John Wiley & Sons, Inc.

2 Page 49 of 51 John Wiley & Sons, Inc.

  Abbreviations used: LCA: Leber Congenital Amaurosis; NPHP: nephronophthisis; JS + RF: Joubert syndrome with associated renal failure.

				Human Mutation Human Mutation		Page 50 of 51
	Page 4: [1] Deleted		Frauke Coppieters		6/14/2010 5:55:00 PM
	c.2118_2122dup	p.Thr709SerfsX9	1
	Unclassified variant			Associated phenotype
	Nucleotide nomenclature	Protein nomenclature	LCA	NPHP	JS +RF	Healthy
	c.1991A>G	p.Asp664Gly			1	1
	c.2717T>G	p.Leu906Trp				1
	c.6684T>G	p.Asn2228Lys				1
	c.4696G>C	p.Ala1566Pro	1
	c.5081T>C	p.Leu1694Pro	1
	c.4661_4663del	p.Glu1554del	1
	c.7311_7313del	p.Lys2437del		1
		F o F o
			r r
			P P
			e e r e e r
					R e v i e w R e v i e w
			John Wiley & Sons, Inc.

Deleted: Table

John Wiley & Sons, Inc.Human Mutation


Konstantinos P Economopoulos
email: economopoulos@sni.gr

Theodoros N Sergentanis

Does race modify the association between CYP1B1 Val432Leu polymorphism and breast cancer risk? A critical appraisal of a recent meta-analysis

Does race modify the association between CYP1B1 Val432Leu polymorphism and breast cancer risk? A critical appraisal of a recent meta-analysis

Konstantinos P. Economopoulos, Theodoros N.

Dear Editor,

We read with great interest the recent meta-analysis by Yao et al. [START_REF] Yao | No association between CYP1B1 Val432Leu polymorphism and breast cancer risk: a meta-analysis involving 40,303 subjects[END_REF], which has reached important conclusions about the association CYP1B1 Val432Leu polymorphism and breast cancer risk. Yao et al. [START_REF] Yao | No association between CYP1B1 Val432Leu polymorphism and breast cancer risk: a meta-analysis involving 40,303 subjects[END_REF] did not demonstrate any significant associations between CYP1B1 Val432Leu polymorphism and breast cancer susceptibility, either at the overall or the race-specific analyses (Asian, Caucasian, African and Mixed).

Nevertheless, close inspection of the studies analyzed by the authors revealed some methodological issues that are worth mentioning and clarifying. Adopting the same search strategy and end-of-search date as Yao et al. [START_REF] Yao | No association between CYP1B1 Val432Leu polymorphism and breast cancer risk: a meta-analysis involving 40,303 subjects[END_REF], we have located three relevant case-control studies in MEDLINE with a total number of 685 breast cancer cases and 931 controls, which have not been included in the meta-analysis [START_REF] Listgarten | Predictive models for breast cancer susceptibility from multiple single nucleotide polymorphisms[END_REF][START_REF] Zimarina | Polymorphisms of CYP1B1 and COMT in breast and endometrial cancer[END_REF][START_REF] Diergaarde | Polymorphisms in genes involved in sex hormone metabolism, estrogen plus progestin hormone therapy use, and risk of postmenopausal breast cancer[END_REF]. Of note, all three casecontrol studies were performed on Caucasian populations. Moreover, a methodological issue seems worth commenting. Specifically, the study by Van Emburgh et al. [START_REF] Van Emburgh | Polymorphisms in CYP1B1, GSTM1, GSTT1 and GSTP1, and susceptibility to breast cancer[END_REF] has provided separate data on Caucasian (381 cases and 412 controls) and African-American (52 cases and 77 controls) subpopulations; worthy of note Yao et al. [START_REF] Yao | No association between CYP1B1 Val432Leu polymorphism and breast cancer risk: a meta-analysis involving 40,303 subjects[END_REF] classified the study as a study on "mixed" populations and did not extract the reported separate data on Caucasian and African-American subgroups. Under the light of the above we have performed the overall meta-analysis, as well as the racespecific analyses in Caucasian, African and mixed populations; the analysis on Asian subjects has been spared, since the issues mentioned above did not pertain to Asian studies.

The overall meta-analysis, as well as the analyses on Caucasian and mixed populations replicated the results reported by Yao et al. [START_REF] Yao | No association between CYP1B1 Val432Leu polymorphism and breast cancer risk: a meta-analysis involving 40,303 subjects[END_REF]; on the other hand a statistically significant finding emerged on African populations. Specifically, at the overall analysis the pooled Odds Ratios (ORs) (95% Confidence Intervals, CI) were 1.021 (0.941-1.109, random effects) for heterozygous and 1.034 (0.930-1.150, random effects) for homozygous Val subjects. Concerning Caucasian subjects, the pooled OR were 1.051 (0.939-1.176, random effects) for heterozygous carriers and 1.038 (0.912-1.180, random effects) for homozygous carriers. Subanalysis on African subjects demonstrated that heterozygous carriers were associated with increased breast cancer risk (pooled OR=1.918, 95% CI:

1.011-3.638, p=0.046, fixed effects), whereas the results concerning homozygous carriers remained at the level of borderline significance (pooled OR= 1.773, 95% CI: 0.947-3.318, p=0.073, fixed effects).

In conclusion, race-specific associations between CYP1B1 Val432Leu polymorphism and breast cancer risk may well exist; further studies on African pop ulations are needed so as to validate the present results in larger sets of case-control studies.