Kinga Bujakowska

Isabelle Audo
email: isabelle.audo@inserm.fr

Saddek Mohand-Saïd

Marie-Elise Lancelot

Aline Antonio

Aurore Germain

Thierry Léveillard

Mélanie Letexier

Jean-Paul Saraiva

Christine Lonjou

Wassila Carpentier

José-Alain Sahel

Shomi S Bhattacharya

Christina Zeitz
email: christina.zeitz@inserm.fr

CRB1 mutations in

Keywords: CRB1, LCA, Retinitis Pigmentosa, rod-cone dystrophy

teaching and research institutions in France or abroad, or from public or private research centers.

Background

Mutations in the CRB1 gene (MIM#: 604210) are associated with variable phenotypes of severe retinal dystrophies, ranging from Leber Congenital Amaurosis (LCA) to rod-cone dystrophy (also called retinitis pigmentosa (RP)) [START_REF] Benayoun | Genetic heterogeneity in two consanguineous families segregating early onset retinal degeneration: the pitfalls of homozygosity mapping[END_REF]Bernal, et al., 2003;Booij, et al., 2005;Clark, et al., 2010;[START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes[END_REF][START_REF] Hollander | CRB1 mutation spectrum in inherited retinal dystrophies[END_REF]den Hollander, et al., 2001a;[START_REF] Hollander | Identification of novel mutations in patients with Leber congenital amaurosis and juvenile RP by genome-wide homozygosity mapping with SNP microarrays[END_REF][START_REF] Hollander | Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12)[END_REF]Galvin, et al., 2005;[START_REF] Gerber | A novel mutation disrupting the cytoplasmic domain of CRB1 in a large consanguineous family of Palestinian origin affected with Leber congenital amaurosis[END_REF][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]Henderson, et al., 2010;Henderson, et al., 2007;Jacobson, et al., 2003;[START_REF] Khaliq | Mutation screening of Pakistani families with congenital eye disorders[END_REF]Li, et al., 2011;Lotery, et al., 2001a;Lotery, et al., 2001b;[START_REF] Riveiro-Alvarez | Molecular analysis of ABCA4 and CRB1 genes in a Spanish family segregating both Stargardt disease and autosomal recessive retinitis pigmentosa[END_REF][START_REF] Seong | Molecular characterization of Leber congenital amaurosis in Koreans[END_REF]Simonelli, et al., 2007;[START_REF] Tosi | Case report: autofluorescence imaging and phenotypic variance in a sibling pair with early-onset retinal dystrophy due to defective CRB1 function[END_REF]Vallespin, et al., 2007;[START_REF] Walia | Visual acuity in patients with Leber's congenital amaurosis and early childhood-onset retinitis pigmentosa[END_REF]Yzer, et al., 2006a;Yzer, et al., 2006b;[START_REF] Zernant | Genotyping microarray (disease chip) for Leber congenital amaurosis: detection of modifier alleles[END_REF]. LCA is a group of the most severe and the earliest occurring retinal dystrophies resulting in congenital blindness [START_REF] Hollander | Leber congenital amaurosis: genes, proteins and disease mechanisms[END_REF]. The onset of the disease occurs at birth and the characteristic features include non-recordable electroretinogram (ERG), nystagmus, sluggish or absent pupillary responses and oculo-digital reflexes, a distinctive eye-rubbing also called the Franschetti sign [START_REF] Hollander | Leber congenital amaurosis: genes, proteins and disease mechanisms[END_REF][START_REF] Franceschetti | Diagnostic and prognostic importance of the electroretinogram in tapetoretinal degeneration with reduction of the visual field and hemeralopia[END_REF][START_REF] Leber | Ueber Retinitis pigmentosa und angeborene Amaurose[END_REF]. RP is a clinically heterogeneous disorder characterised by a progressive degeneration of the photoreceptors and leading to a visual impairment of variable severity that can end in complete blindness. The disease onset is highly variable: it may commence in the first decade of life or much later. There is a considerable clinical overlap between LCA and early-onset RP and in some cases/reports the diagnosis is ambiguous. Early-onset RP, however, is considered as a relatively milder form, where patients do not have a congenital onset of visual impairment.

LCA and RP resulting from CRB1 mutations may be accompanied by specific fundus features: preservation of the para-arteriolar retinal pigment epithelium (PPRPE) (Bernal, et al., 2003;[START_REF] Hollander | CRB1 mutation spectrum in inherited retinal dystrophies[END_REF][START_REF] Hollander | Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12)[END_REF][START_REF] Heckenlively | Preserved para-arteriole retinal pigment epithelium (PPRPE) in retinitis pigmentosa[END_REF]Henderson, et al., 2010;[START_REF] Khaliq | Mutation screening of Pakistani families with congenital eye disorders[END_REF]Simonelli, et al., 2007;Yzer, et al., 2006b) and retinal telangiectasia with exudation (also referred to as Coats-like vasculopathy) [START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes[END_REF][START_REF] Hollander | CRB1 mutation spectrum in inherited retinal dystrophies[END_REF]den Hollander, et al., 2001a;Henderson, et al., 2010;Yzer, et al., 2006b). PPRPE is characterized by a relative preservation of retinal pigment epithelium (RPE) adjacent to retinal arterioles despite a panretinal RPE degeneration [START_REF] Heckenlively | Preserved para-arteriole retinal pigment epithelium (PPRPE) in retinitis pigmentosa[END_REF]. This is, however, not consistent in CRB1-associated RP and the absence of PPRPE in a severe RP should not exclude CRB1 as a potential causal gene (Lotery, et al., 2001b).

Retinal telangiectasia is a condition of abnormally permeable blood vessels, leading to exudation and retinal detachment [START_REF] Cahill | Classification of the spectrum of Coats' disease as subtypes of idiopathic retinal telangiectasis with exudation[END_REF]. Some patients with CRB1 mutations show macular atrophy (Henderson, et al., 2010), similar features were found for other LCA causing genes (GUCY2D MIM#:600179, AIPL1 MIM#:604392 and RPGRIP1 MIM#:605446), which lead to classification of LCA into cone-rod LCA and rod-cone LCA [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]. Patients with CRB1 mutations belong to both categories. Predisposition of the CRB1 patients to keratoconus [START_REF] Mckibbin | Genotype-phenotype correlation for leber congenital amaurosis in Northern Pakistan[END_REF][START_REF] Mcmahon | CRB1 gene mutations are associated with keratoconus in patients with leber congenital amaurosis[END_REF] and implication for pigmented paravenous chorioretinal atrophy [START_REF] Mckay | Pigmented paravenous chorioretinal atrophy is associated with a mutation within the crumbs homolog 1 (CRB1) gene[END_REF] and nanophthalmos [START_REF] Zenteno | Homozygosity mapping identifies the Crumbs homologue 1 (Crb1) gene as responsible for a recessive syndrome of retinitis pigmentosa and nanophthalmos[END_REF] have also been reported.

CRB1 is a human homologue of the Drosophila melanogaster gene coding for protein crumbs (crb) and it is expressed in the retina and the brain (den [START_REF] Hollander | Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12)[END_REF]. CRB1 consists of 12 exons and exhibits alternative splicing at the 3' end, yielding two proteins of 1376 and 1406 amino acids (den Hollander, et al., 2001b).

Both proteins contain 19 EGF-like domains, three laminin AG-like domains and a signal peptide sequence. In addition, the longer isoform contains transmembrane and cytoplasmic domains (den Hollander, et al., 2001b;[START_REF] Gosens | Composition and function of the Crumbs protein complex in the mammalian retina[END_REF]. The cytoplasmic domain includes conserved FERM and PDZ binding motifs, through which CRB1 participates in the formation of adherens junction and links to the actin cytoskeleton [START_REF] Gosens | Composition and function of the Crumbs protein complex in the mammalian retina[END_REF].

In Drosophila, crb determines the polarity of the embryonic epithelium and peripheral neurons; it is important for the maintenance of zonula adherens (ZA) and it is localized in the apical membrane [START_REF] Tepass | crumbs encodes an EGF-like protein expressed on apical membranes of Drosophila epithelial cells and required for organization of epithelia[END_REF]. In the mouse retina, Crb1 is present in the apical membranes of the epithelial cells, in Muller cells and in photoreceptor inner segments, where it concentrates in the vicinity of the outer limiting membrane [START_REF] Hollander | Isolation of Crb1, a mouse homologue of Drosophila crumbs, and analysis of its expression pattern in eye and brain[END_REF][START_REF] Mehalow | CRB1 is essential for external limiting membrane integrity and photoreceptor morphogenesis in the mammalian retina[END_REF][START_REF] Pellikka | Crumbs, the Drosophila homologue of human CRB1/RP12, is essential for photoreceptor morphogenesis[END_REF][START_REF] Van De Pavert | Crumbs homologue 1 is required for maintenance of photoreceptor cell polarization and adhesion during light exposure[END_REF]. A similar distribution was found in the human retina (van [START_REF] Van De Pavert | Crumbs homologue 1 is required for maintenance of photoreceptor cell polarization and adhesion during light exposure[END_REF]. Crumbs and its mouse homolog Crb1 is involved in the photoreceptor morphogenesis [START_REF] Pellikka | Crumbs, the Drosophila homologue of human CRB1/RP12, is essential for photoreceptor morphogenesis[END_REF][START_REF] Tepass | crumbs encodes an EGF-like protein expressed on apical membranes of Drosophila epithelial cells and required for organization of epithelia[END_REF].

Analysis of the naturally occurring Crb1 rd8 mouse mutant, suggests a developmental defect of the retina, where disruption of the outer limiting membrane and formation of retinal folds (pseudorosettes) are observed [START_REF] Mehalow | CRB1 is essential for external limiting membrane integrity and photoreceptor morphogenesis in the mammalian retina[END_REF]. Disorganization of the retinal layers was also noted in other Crb1 mouse models (van [START_REF] Van De Pavert | Crumbs homologue 1 is required for maintenance of photoreceptor cell polarization and adhesion during light exposure[END_REF][START_REF] Van De Pavert | A single amino acid substitution (Cys249Trp) in Crb1 causes retinal degeneration and deregulates expression of pituitary tumor transforming gene Pttg1[END_REF]. These findings are in accordance with clinical features of the patients carrying CRB1 mutations, whose retinas are thickened and show an altered laminar organization, resembling an immature normal retina (Jacobson, et al., 2003). The latter further supports the importance of CRB1 in the development of the retina.

This study presents an overview of the previously published CRB1 variants and novel mutations identified in a French cohort of simplex and autosomal recessive RP (arRP) patients. Based on the available genetic and phenotypic data from the literature and on our original findings, we classify all variants into one of the three groups (likely pathogenic, unclassified variants and unlikely pathogenic, Supp. Tables S1-S3). We discuss the clinical variability of patients harboring CRB1 mutations and analyse the phenotype-genotype correlation of likely pathogenic changes.

Identification of novel mutations in the French cohort is described (Supp. Methods and Results) and precise clinical characterisation is given.

Novel CRB1 Variants

Eleven unrelated patients with ar or isolated RP in the French cohort carried likely pathogenic variants of CRB1 (Table1). Seven mutations were novel: three missense changes (p.Ser740Phe, p.Tyr1198Cys and p.Cys1223Ser), one nonsense mutation (p.Cys423*), one in-frame deletion (p.Asn789del) and two frameshift deletions (p.Leu655Trpfs*10, p.Ser1220Asnfs*62) (Table 1). Mutations identified in this study were not present in the SNP databases nor listed as non-pathogenic variants in the literature. None of the novel mutations was present in at least 362 control alleles and the mutations co-segregated in available family members (Supp. Figure S1). In all but one patient (547) two mutated CRB1 alleles were found.

The three novel missense mutations are in the conserved domains of the CRB1 protein. The p.Ser740Phe exchange replaces a highly conserved serine in the second laminin AG-like domain, the p.Tyr1198Cys mutation replaces a conserved tyrosine with a cysteine in the 16 th calcium binding EGF-like domain and the p.Cys1223Ser is a replacement of a conserved cysteine with a serine in the 17 th calcium binding EGF-like domain (Figure 1). The in-frame deletion p.Asn789del is also located in the second laminin AG-like domain. Other novel mutations (p.Cys423*, p.Leu655Trpfs*10, p.Ser1220Asnfs*62) result in premature stop codons, which most likely lead to nonsense mediated decay [START_REF] Chang | The nonsense-mediated decay RNA surveillance pathway[END_REF] and therefore these alleles are considered as null alleles. Five novel mutations are within exons 7 and 9, which are the most frequently mutated (Figure 1).

Clinical Characterisation of Patients with CRB1 Mutations

Clinical findings of French patients with CRB1 mutations are summarized in Tables 2 and3. The average age at time of diagnosis was 17. Visual acuity was decreased in all patients ranging from 20/50 to light perception with no clear correlation with age or duration of the disease. Hyperopia was noted for 6/11 patients including three for whom spherical equivalent was equal or above +5 diopters. Night blindness was present in all patients but three, for whom a decrease of central vision and photophobia dominated. None of the patients had nystagmus. Most patients (9/11) had a clear lens; in the remaining two, one had undergone cataract surgery and one had significant lens opacities. These two patients were over 40 years of age. Two patterns of fundus pigmentary changes were present in this cohort: 7/11 had typical bone spicule-shaped pigment migration within the peripheral retina whereas 4/11 had widespread clumped pigmentary changes of nummular appearance at the level of the retinal pigment epithelium (Figure 2). Clumped pigmentation is therefore highly suggestive of CRB1 mutations but it is not specific since it has also been associated with mutations in NR2E3 [START_REF] Schorderet | NR2E3 mutations in enhanced S-cone sensitivity syndrome (ESCS), Goldmann-Favre syndrome (GFS), clumped pigmentary retinal degeneration (CPRD), and retinitis pigmentosa (RP)[END_REF][START_REF] Sharon | Shared mutations in NR2E3 in enhanced S-cone syndrome, Goldmann-Favre syndrome, and many cases of clumped pigmentary retinal degeneration[END_REF], NRL [START_REF] Nishiguchi | Recessive NRL mutations in patients with clumped pigmentary retinal degeneration and relative preservation of blue cone function[END_REF] or TULP1 [START_REF] Mataftsi | Novel TULP1 mutation causing leber congenital amaurosis or early onset retinal degeneration[END_REF]. None of the patients displayed preservation of the para-arteriolar retinal pigment epithelium as previously described in association with CRB1 mutations (Bernal, et al., 2003;[START_REF] Hollander | CRB1 mutation spectrum in inherited retinal dystrophies[END_REF][START_REF] Hollander | Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12)[END_REF][START_REF] Heckenlively | Preserved para-arteriole retinal pigment epithelium (PPRPE) in retinitis pigmentosa[END_REF]Henderson, et al., 2010;[START_REF] Khaliq | Mutation screening of Pakistani families with congenital eye disorders[END_REF]Simonelli, et al., 2007;Yzer, et al., 2006b). In addition, none of the patients displayed Coats-like changes in the periphery. All patients had macular involvement. Six of the patients displayed cystoid macular edema whereas the other five had macular thinning with loss of the outer retinal layers and corresponding loss of autofluorescence (Figure 2). Color vision was normal in four patients or showed either tritan deficit or a dyschromatopsia with no clear axis when visual acuity allowed color vision testing. Full field electroretinogram showed severe generalized retinal dysfunction with no detectable responses in all patients except three for whom some residual rod and cone function was detectable. Among those three, the best responses on ERG were obtained in the youngest patients. Residual responses on ERG were correlated with better preservation of the visual field.

All patients displayed severe retinal involvement with early macular changes, half of them had cystoid macular edema, a higher percentage than the usually reported prevalence of about 30% in overall RP [START_REF] Hajali | The prevalence of cystoid macular oedema in retinitis pigmentosa patients determined by optical coherence tomography[END_REF]. This higher prevalence could at least be in part related to vascular abnormalities with Coats-like changes encountered in patients with CRB1 mutations [START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes[END_REF][START_REF] Hollander | CRB1 mutation spectrum in inherited retinal dystrophies[END_REF]den Hollander, et al., 2001a;Henderson, et al., 2010;Yzer, et al., 2006b).

Alternatively, these changes could be related to abnormal laminar structure associated with CRB1-mutations (Jacobson, et al., 2003). None of our patients developed Coatslike changes or para-arteriolar retinal pigment epithelium suggesting that these changes are not consistant in CRB1-related RP (Lotery, et al., 2001b). Four subjects displayed clumped retinopathies reinforcing that CRB1 should be considered as a potential causal gene for this specific phenotype along with NR2E3 [START_REF] Sharon | Shared mutations in NR2E3 in enhanced S-cone syndrome, Goldmann-Favre syndrome, and many cases of clumped pigmentary retinal degeneration[END_REF] or NRL [START_REF] Nishiguchi | Recessive NRL mutations in patients with clumped pigmentary retinal degeneration and relative preservation of blue cone function[END_REF].

CRB1 Variants and Their Classification

Over 240 patients with CRB1 mutations and more than 150 gene variants have been described in the literature [START_REF] Benayoun | Genetic heterogeneity in two consanguineous families segregating early onset retinal degeneration: the pitfalls of homozygosity mapping[END_REF]Bernal, et al., 2003;Booij, et al., 2005;Clark, et al., 2010;[START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes[END_REF][START_REF] Hollander | CRB1 mutation spectrum in inherited retinal dystrophies[END_REF]den Hollander, et al., 2001a;[START_REF] Hollander | Identification of novel mutations in patients with Leber congenital amaurosis and juvenile RP by genome-wide homozygosity mapping with SNP microarrays[END_REF][START_REF] Hollander | Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12)[END_REF]Galvin, et al., 2005;[START_REF] Gerber | A novel mutation disrupting the cytoplasmic domain of CRB1 in a large consanguineous family of Palestinian origin affected with Leber congenital amaurosis[END_REF][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]Henderson, et al., 2010;Henderson, et al., 2007;Jacobson, et al., 2003;[START_REF] Khaliq | Mutation screening of Pakistani families with congenital eye disorders[END_REF]Li, et al., 2011;Lotery, et al., 2001a;Lotery, et al., 2001b;[START_REF] Riveiro-Alvarez | Molecular analysis of ABCA4 and CRB1 genes in a Spanish family segregating both Stargardt disease and autosomal recessive retinitis pigmentosa[END_REF][START_REF] Seong | Molecular characterization of Leber congenital amaurosis in Koreans[END_REF]Simonelli, et al., 2007;[START_REF] Tosi | Case report: autofluorescence imaging and phenotypic variance in a sibling pair with early-onset retinal dystrophy due to defective CRB1 function[END_REF]Vallespin, et al., 2007;Yzer, et al., 2006a;Yzer, et al., 2006b;[START_REF] Zenteno | Homozygosity mapping identifies the Crumbs homologue 1 (Crb1) gene as responsible for a recessive syndrome of retinitis pigmentosa and nanophthalmos[END_REF][START_REF] Zernant | Genotyping microarray (disease chip) for Leber congenital amaurosis: detection of modifier alleles[END_REF]. The most frequently occurring of the known mutations is the p.Cys948Tyr in exon 9 (96 alleles reported, 24% of known CRB1 mutations) (Bernal, et al., 2003;Booij, et al., 2005;Clark, et al., 2010;[START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes[END_REF][START_REF] Hollander | CRB1 mutation spectrum in inherited retinal dystrophies[END_REF]den Hollander, et al., 2001a;[START_REF] Hollander | Identification of novel mutations in patients with Leber congenital amaurosis and juvenile RP by genome-wide homozygosity mapping with SNP microarrays[END_REF][START_REF] Hollander | Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12)[END_REF]Galvin, et al., 2005;[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]Henderson, et al., 2010;Henderson, et al., 2007;Jacobson, et al., 2003;Lotery, et al., 2001a;[START_REF] Riveiro-Alvarez | Molecular analysis of ABCA4 and CRB1 genes in a Spanish family segregating both Stargardt disease and autosomal recessive retinitis pigmentosa[END_REF][START_REF] Tosi | Case report: autofluorescence imaging and phenotypic variance in a sibling pair with early-onset retinal dystrophy due to defective CRB1 function[END_REF]Vallespin, et al., 2007;Yzer, et al., 2006a;[START_REF] Zernant | Genotyping microarray (disease chip) for Leber congenital amaurosis: detection of modifier alleles[END_REF]. In general most of the mutations are in exons 9 (41%) and 7 (27%), therefore as a screening strategy these exons can be tested in the first instance (Figure 1, Supp. Table S1). Exons 7 and 9 encode second and third laminin AG-like domains respectively, implying that these domains are particularly important for CRB1 function. Missense mutations constitute 66% of all known mutations, the remaining being frameshift, truncation and splice site mutations.

We have attempted to classify all the reported mutations in three groups: 1) likely pathogenic, 2) unclassified variants, 3) unlikely pathogenic. This classification was based on the genetic data available from the literature, amino acid conservation and bioinformatic pathogenicity prediction tools (Supp. Tables S1-S3). An important criterion was the presence of two mutant alleles and co-segregation in the family.

Approximately 30% of cases were reported with only one mutant allele, assuming that the second mutation is within the intronic region. For these patients however, one cannot exclude the possibility that there is another molecular cause of the pathology.

The lack of the second mutant CRB1 allele is sometimes explained by a digenic inheritance, however so far it has not been proven by co-segregation analysis (Li, et al., 2011;Vallespin, et al., 2007).

Pathogenicity is easier to asses in deletions and frameshift variants than in the case of missense changes, hence the importance of the bioinformatic analysis of the pathogenicity, amino acid conservation and functional analysis of the variants. On this basis we have not considered two changes identified in our cohort as pathogenic (p.Gly959Ser and p.Ala1354Thr) [START_REF] Hollander | CRB1 mutation spectrum in inherited retinal dystrophies[END_REF]den Hollander, et al., 2001a)). The respective patients did not carry a second CRB1 mutation and we did not consider the p.Gly959Ser and p.Ala1354Thr substitutions as likely pathogenic, based on poor conservation of the residues and low pathogenicity predictions using online bioinformatic tools: PolyPhen-2 and SIFT (Supp. Tables S2 andS3). One report suggests involvement of CRB1 in autosomal dominant pigmented paravenous chorioretinal atrophy [START_REF] Mckay | Pigmented paravenous chorioretinal atrophy is associated with a mutation within the crumbs homolog 1 (CRB1) gene[END_REF], though the reported mutation p.Val162Met has a questionable pathogenicity, since valine is not conserved and methionine is present in this position in other mammals (Supp. Table S2).

Prevalence

In the investigated cohort, at least 2.5% of arRP patients carry CRB1 gene defects, which lies within the previously published range of 0-6.5% (Bernal, et al., 2003;[START_REF] Hollander | CRB1 mutation spectrum in inherited retinal dystrophies[END_REF]Vallespin, et al., 2007), or 2.7% after cohort averaging (Table 4). The high preponderance of novel CRB1 mutations in our cohort suggests, however, that probably more arRP patients carry CRB1 pathogenic defects, which are novel and therefore undetectable by arRP microarray. Much higher prevalence is observed in LCA/EORD cohorts and RP with additional features like PPRPE and retinal telangiectasia, representing 10.1%, 74.1%, 53.3% respectively in averaged cohorts (Table 4) (Bernal, et al., 2003;[START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes[END_REF][START_REF] Hollander | CRB1 mutation spectrum in inherited retinal dystrophies[END_REF]den Hollander, et al., 2001a;[START_REF] Hollander | Identification of novel mutations in patients with Leber congenital amaurosis and juvenile RP by genome-wide homozygosity mapping with SNP microarrays[END_REF][START_REF] Hollander | Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12)[END_REF][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]Henderson, et al., 2010;Henderson, et al., 2007;Lotery, et al., 2001a;[START_REF] Seong | Molecular characterization of Leber congenital amaurosis in Koreans[END_REF]Simonelli, et al., 2007;Vallespin, et al., 2007;[START_REF] Walia | Visual acuity in patients with Leber's congenital amaurosis and early childhood-onset retinitis pigmentosa[END_REF].

Genotype-Phenotype Correlation

We were not able to establish a clear genotype/phenotype correlation for our cohort, which might be due to the small number of patients with CRB1 mutations and their variable phenotype. In addition, the nature of existing published data makes it difficult to correlate the recurring CRB1 mutations with different phenotypes for a number of reasons. First, the phenotyping of patients is complex and distinguishing between early-onset RP and LCA is often arbitrary and depends on the guidelines of a particular clinical center. Second, precise clinical data is often omitted in the publications and therefore it is difficult to adjust for these diagnostic differences in a cross-paper analysis. Despite these inconsistencies, we attempted to analyse data from previous reports in order to find the relationship between the CRB1 variants and the clinical features of respective patients. In this meta-analysis we used 171 patients, who carried two likely pathogenic mutations in trans [START_REF] Benayoun | Genetic heterogeneity in two consanguineous families segregating early onset retinal degeneration: the pitfalls of homozygosity mapping[END_REF]Bernal, et al., 2003;Booij, et al., 2005;Clark, et al., 2010;[START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes[END_REF][START_REF] Hollander | CRB1 mutation spectrum in inherited retinal dystrophies[END_REF]den Hollander, et al., 2001a;[START_REF] Hollander | Identification of novel mutations in patients with Leber congenital amaurosis and juvenile RP by genome-wide homozygosity mapping with SNP microarrays[END_REF][START_REF] Hollander | Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12)[END_REF]Galvin, et al., 2005;[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]Henderson, et al., 2010;Henderson, et al., 2007;Jacobson, et al., 2003;[START_REF] Khaliq | Mutation screening of Pakistani families with congenital eye disorders[END_REF]Li, et al., 2011;Lotery, et al., 2001a;Lotery, et al., 2001b;[START_REF] Mckibbin | Genotype-phenotype correlation for leber congenital amaurosis in Northern Pakistan[END_REF][START_REF] Riveiro-Alvarez | Molecular analysis of ABCA4 and CRB1 genes in a Spanish family segregating both Stargardt disease and autosomal recessive retinitis pigmentosa[END_REF][START_REF] Seong | Molecular characterization of Leber congenital amaurosis in Koreans[END_REF]Simonelli, et al., 2007;[START_REF] Tosi | Case report: autofluorescence imaging and phenotypic variance in a sibling pair with early-onset retinal dystrophy due to defective CRB1 function[END_REF]Vallespin, et al., 2007;Yzer, et al., 2006a). Combination of two mutant alleles was analysed in relation to clinical characteristics of the published cases. Based on the reports we distinguished the following phenotypes: LCA, early onset retinal degeneration (EORD), RP, presence of PPRPE and Coats-like vasculopathy. The mutations were classed as null mutations (all mutations leading to a premature stop codon) or as variants leading to an altered protein (missense and in frame deletions).

The likely pathogenic mutations were plotted on a graph, where affected codons on allele 1 and allele 2 served as coordinates (codon 0 was assigned to null mutations).

The results show that we cannot assign a specific allele combination to a particular phenotype, e.g. homozygous null alleles or homozygous p.Cys948Tyr alleles are found in LCA, EORD and RP patients (Figure 3 A). Null alleles are however more frequent in LCA cohorts (Figure 3 B) as previously suggested [START_REF] Hollander | CRB1 mutation spectrum in inherited retinal dystrophies[END_REF]. The presence/absence of PPRPE or Coats-like vasculopathy did not reveal a particular mutation pattern (Figure 3 C). These findings suggest the involvement of additional modifying factors (genetic and/or environmental), which are responsible for the modulation of the phenotype in patients harboring CRB1 mutations.

Future Directions

The above analysis of the phenotype-genotype correlation suggests that the disease severities associated with CRB1 mutations are in fact a continuum of the same clinical entity with possible additional modifying factors influencing disease onset and progression. There is increasing evidence of the involvement of multiple alleles in the patient's phenotype, as has been shown for the Bardet-Biedl patients [START_REF] Katsanis | Triallelic inheritance in Bardet-Biedl syndrome, a Mendelian recessive disorder[END_REF] and more recently for a PRPH2-associated macular dystrophy family, where the phenotype has been modulated by additional heterozygous mutations in ABCA4 (MIM#: 601691) and ROM1 (MIM#: 180721) [START_REF] Poloschek | ABCA4 and ROM1: implications for modification of the PRPH2-associated macular dystrophy phenotype[END_REF]. It is likely that the new next generation sequencing (NGS) technology will help to shed light on the potential genetic modifiers that influence disease phenotype. One has, however, to analyse the data with caution since NGS will reveal large numbers of polymorphic changes, which do not modulate the disease. The potential new modifying changes will have to be confirmed by appropriate genetic and functional analysis. The certainty of the molecular cause of a disease is particularly important in the era of gene therapy trials. Genetic treatment of recessive disorders should not be undertaken before obtaining proof that both alleles of a given gene are dysfunctional.

In-depth genetic analysis, as presented here, is necessary to provide a basis for conducting such therapies. (Bernal, et al., 2003;[START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes[END_REF][START_REF] Hollander | CRB1 mutation spectrum in inherited retinal dystrophies[END_REF]den Hollander, et al., 2001;[START_REF] Hollander | Identification of novel mutations in patients with Leber congenital amaurosis and juvenile RP by genome-wide homozygosity mapping with SNP microarrays[END_REF][START_REF] Hollander | Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12)[END_REF][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]Henderson, et al., 2010;Henderson, et al., 2007;Li, et al., 2011;Lotery, et al., 2001;[START_REF] Seong | Molecular characterization of Leber congenital amaurosis in Koreans[END_REF]Simonelli, et al., 2007;Vallespin, et al., 2007;[START_REF] Walia | Visual acuity in patients with Leber's congenital amaurosis and early childhood-onset retinitis pigmentosa[END_REF] RP 2.7% 4 5 335 (Bernal, et al., 2003;[START_REF] Hollander | CRB1 mutation spectrum in inherited retinal dystrophies[END_REF]Vallespin, et al., 2007 * The average prevalence was calculated on the basis of all the published reports indicating phenotypes of patients with CRB1 mutations and the size of screened cohorts.

SUPPORTING MATERIAL

Methods and Results

Clinical assessment

Patients with a provisional diagnosis of arRP were collected and clinically examined in the Clinical Investigating Centre of the Quinze-Vingts Hospital.

Informed consent was obtained from each patient and normal controls after explanation of the study and its potential outcome. The study protocol adhered to the tenets of the Declaration of Helsinki and was approved by the local ethics committee.

Each patient underwent full ophthalmic examination with clinical assessment as described earlier. [START_REF] Audo | EYS is a major gene for rod-cone dystrophies in France[END_REF]. For additional family members who could not come to our centre for examination, ophthalmic records were obtained from local ophthalmologists.

Mutation detection by arRP microarray

Total genomic DNA was extracted from peripheral blood leucocytes according to manufacturer's recommendation (Puregen Kit, Qiagen, Courtaboeuf, France). The DNAs of 400 index patients were analyzed for known mutations by microarray analysis on a commercially available chip (arRP, ASPER Ophthalmics, Tartu, Estonia). Mutations identified by this approach were validated by direct Sanger sequencing. In cases where only one heterozygous mutation was detected, the second mutation was identified by direct sequencing of all exons and flanking intronic sequences of CRB1 (NM_201253.2; including alternative transcript AF154671.1).

Out of 400 index patients nine probands were found to have CRB1 mutations on the microarray. Two patients were homozygous and two other compound heterozygous for known mutations. Four patients were heterozygous for one known mutation and one patient showed an unexpected event in exon 6 of CRB1. 1).

Homozygosity mapping

One consanguineous family (F709), excluded for known mutations by the first screening approach, was analysed using a 700K SNP microarray (HumanOmniExpress, Illumina, Eindhoven, The Netherlands). The SNP genotypes were analysed using commercially available software (GenomeStudio, Illumina, Eindhoven, The Netherlands) according to the protocols provided by Illumina. In the initial analysis, 686389 SNPs passed quality control. The homozygous regions were found through a web-based tool HomozygosityMapper (http://www.homozygositymapper.org/) [START_REF] Seelow | HomozygosityMapper--an interactive approach to homozygosity mapping[END_REF].

The analysis revealed eight significant homozygous regions on chromosome 1 (16, 17 and 53 Mb), chromosome 4 (29 Mb), chromosome 6 (16 and 20 Mb) and chromosome 12 (13 and 56 Mb). These homozygous regions contained ten known retinopathy genes: (ABCA4, PRPF3, SEMA4A, CRB1, CC2D2A, BBS7, BBS12, PROM1, BBS10, CEP290) of which CRB1 was the most promising candidate as suggested by the patient's phenotype. CRB1 was located in a 17 Mb homozygous region on chromosome 1, which was the 4 th largest homozygous region. Direct sequencing of CRB1 revealed a novel homozygous deletion-insertion in exon 9 (c.3659_3660delinsA, p.Ser1220Asnfs*62) (Table 1).

Next generation sequencing (NGS)

One consanguineous family was investigated by NGS using a custom-made oligonucleotide library targeting 177 known genes underlying retinal disorders The first screening criteria applied to the index patient form the consanguineous family were absence of the variant in dbSNP databases and homozygous appearance.

This initial screen resulted in three homozygous mutations, of which p.Ser740Phe exchange in CRB1 was the most convincing (Table 1 in the main test). This mutation was confirmed by Sanger sequencing and by performing cosegregation analysis in the family members (Figure 1). More details on data analysis from the NGS study of retinal genes are published elsewhere (Audo et al., 2011 "Application of nextgeneration-sequencing (NGS) allows novel genotype-phenotype correlations of retinal diseases").

Sanger sequencing

For Sanger sequencing, CRB1 gene (CRB1 RefSeq NM_201253) was PCR amplified in 15 fragments using oligonucl eotides flanking the exons and a polymerase (HotFire, Solis Biodyne, Estonia) in the presence of 1.

Mutation nomenclature and assessment of the pathogenicity of mutations

Nucleotide numbering is based on cDNA sequence of CRB1 (Ref.

NM_201253.2) where A of the ATG initiation codon is 1. To evaluate the pathogenicity of the novel changes we applied the following criteria: 1) stop/frameshift mutations are most likely disease causing; 2) cosegregation in the family; 3) absence in control samples; 4) for missense mutations and in-frame deletions, amino acid conservation was studied in the UCSC Genome Browser using 27 species belonging to different evolutionary branches (Human, Chimp, Gorilla, Rhesus, Tarsier, Mouse lemur, Bushbaby, Tree shrew, Mouse, Squirrel, Rabbit, Cow, Horse, Cat, Dog, Hedgehog, Elephant, Sloth, Wallaby, Opossum, Platypus, Chicken, Lizard, X.tropicalis, Tetraodon, Stickleback and Zebrafish); if the amino acid residue did not change throughout the species it was considered as "highly conserved"; if a change was seen in fewer than five species and not in the primates then it was considered as "moderately conserved"; if a change was present in 5-7, it was considered as "weakly conserved"; otherwise the amino acid residue was considered as "not conserved"; 5) pathogenicity predictions with bioinformatic tools (PolyPhen-2, Polymorphism Phenotyping, http://genetics.bwh.harvard.edu/pph2/ (Adzhubei, et al.), and SIFT, Sorting Intolerant From Tolerant; http://blocks.fhcrc.org/sift/SIFT.html [START_REF] Ng | SIFT: Predicting amino acid changes that affect protein function[END_REF]); 6) presence of the second mutant allele. These criteria were applied to the mutations found in the patients described in this study as well as for the previously published mutations. All the variants were classified into three groups: likely pathogenic; unclassified variants, unlikely pathogenic. This classification is only indicative and has been based on the above criteria. (Henderson, et al., 2010;[START_REF] Khaliq | Mutation screening of Pakistani families with congenital eye disorders[END_REF] 7 c.2548_2551del GGCT p.Gly850Valfs*5 LamAG 2 protein truncation, NMD --2 LCA unknown second allele (Galvin, et al., 2005;Lotery, et al., 2001a (Clark, et al., 2010;[START_REF] Hollander | CRB1 mutation spectrum in inherited retinal dystrophies[END_REF]Henderson, et (den Hollander, et al., 2001a;Galvin, et al., 2005;[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]Lotery, et al., 2001a;Vallespin, et al., 2007b;[START_REF] Zernant | Genotyping microarray (disease chip) for Leber congenital amaurosis: detection of modifier alleles[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[END_REF][START_REF] Hollander | Identification of novel mutations in patients with Leber congenital amaurosis and juvenile RP by genome-wide homozygosity mapping with SNP microarrays[END_REF]Yzer, et al., 2006) Galvin, et al., 2005;Henderson, et al., 2010;Lotery, et al., 2001a) [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]Henderson, et al., 2010;Henderson, et al., 2007;Li, et al., 2011) (Clark, et al., 2010;[START_REF] Hollander | CRB1 mutation spectrum in inherited retinal dystrophies[END_REF][START_REF] Hollander | Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12)[END_REF][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]Henderson, et al., 2010;Simonelli, et al., 2007;Yzer, et al., 2006) (Bernal, et al., 2003;Jacobson, et al., 2003;[START_REF] Tosi | Case report: autofluorescence imaging and phenotypic variance in a sibling pair with early-onset retinal dystrophy due to defective CRB1 function[END_REF]Vallespin, et al., 2007b) [START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes[END_REF][START_REF] Hollander | CRB1 mutation spectrum in inherited retinal dystrophies[END_REF]den Hollander, et al., 2001b;[START_REF] Hollander | Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12)[END_REF]Galvin, et al., 2005;[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]Henderson, et al., 2010;Henderson, et al., 2007;Jacobson, et al., 2003;Lotery, et al., 2001a;Vallespin, et al., 2007b (Booij, et al., 2005;Clark, et al., 2010;[START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes[END_REF][START_REF] Hollander | CRB1 mutation spectrum in inherited retinal dystrophies[END_REF]den Hollander, et al., 2001a;den Hollander, et al., 2001b;Galvin, et al., 2005;Henderson, et al., 2010;Henderson, et al., 2007;Jacobson, et al., 2003;Simonelli, et al., 2007;Yzer, et al no cosegregation (Bernal, et al., 2003;den Hollander, et al., 2001a;Lotery, et al., 2001a;Simonelli, et al., 2007;Vallespin, et al present in control alleles, no cosegregation and no second CRB1 mutation found (Bernal, et al., 2003;Lotery, et al., 2001a;Vallespin, et al., 2007b;[START_REF] Zernant | Genotyping microarray (disease chip) for Leber congenital amaurosis: detection of modifier alleles[END_REF] Hollander, et al., 2004;Vallespin, et al., 2007b;[START_REF] Zernant | Genotyping microarray (disease chip) for Leber congenital amaurosis: detection of modifier alleles[END_REF] (Bernal, et al., 2003;den Hollander, et al., 2001a;Lotery, et al., 2001a;Vallespin, et al., 2007b) Nucleotide

Supplement

Figure 1 .

 1 Figure 1. Distribution of CRB1 mutations in the gene and protein. A) Nucleotide

Figure 2 .

 2 Figure 2. Fundus color photographs and Optical Coherence Tomography (OCT).

Figure 3 .

 3 Figure 3. Genotype-phenotype correlation of patients with CRB1 mutations. A)

  Figure1

(

  http://www.sph.uth.tmc.edu/retnet/sum-dis.htm, October 2010) and additional candidate genes(Audo et al., 2011 "Application of next-generation-sequencing (NGS) allows novel genotype-phenotype correlations of retinal diseases"). A custom-made SureSelect oligonucleotide probe library was designed to capture the exons according to Agilent's recommendations, using the eArray web-based probe design tool (https://earray.chem.agilent.com/earray). The following parameters were chosen for probe design: 120 bp length, 3x probe-tiling frequency, 20 bp overlap allowed in avoided region and exclusion of repetitive DNA sequences identified by implementing eArray's RepeatMasker program. A total of 27 430 probes, covering 1 177 Mb, were designed and synthesized by Agilent Technologies (Santa Clara, CA, USA). Sequence capture, enrichment, and elution were performed according to the manufacturer's instructions (SureSelect, Agilent). Briefly, 3 µg of each genomic DNA were fragmented by sonication and purified to yield fragments of 150-200 bp. Pairedend adaptor oligonucleotides from Illumina were ligated on repaired DNA fragments, which were then purified and enriched by 6 PCR cycles. 500ng of the purified libraries were hybridized to the SureSelect oligo probe capture library for 24h. After hybridization, washing, and elution, the eluted fraction was PCR-amplified with 14 cycles, purified and quantified by qPCR to obtain sufficient DNA template for downstream applications. Each eluted-enriched DNA sample was then sequenced on an Illumina GAIIx as paired-end 75 bp reads. Image analysis and base calling was performed using Illumina Real Time Analysis (RTA) Pipeline version 1.10 with default parameters. Sequence reads were aligned to the reference human genome (UCSC hg19) using commercially available software (CASAVA1.7, Illumina) and the ELANDv2 alignment algorithm. Genetics variation annotation was performed using the in-house pipeline, which consisted of gene annotation (RefSeq), detection of known polymorphisms (dbSNP 131, 1000 Genome) followed by a mutation characterization (exonic, intronic, silent, nonsense etc.). For each position, the exomic frequencies (homozygous and heterozygous) were determined from all the exomes already sequenced by Integragen, and the exome results provided by HapMap project.

Figure S1 .

 S1 Cosegregation analysis of CRB1 mutations in nine arRP families. Circles indicate females and squares males, the filled symbols represent affected individuals and the empty symbols denote healthy family members. Arrows indicate index patients and the question mark denotes an unknown allele.

  Novel mutations are presented in bold. Nucleotide numbering is based on cDNA sequence from the Ref. NM_201253.2, where A of the ATG initiation codon is 1. Lam AG -Laminin AG like domain; TMtransmembrane; Ccytoplasmic; LCA -Leber congenital amaurosis; RPretinitis pigmentosa; EORDearly onset retinal dystrophy; PPRPEpreservation of para-arteriolar retinal pigment epithelium. For PolyPhen-2 the HumVar value was taken, which is preferred for the diagnostic of human Mendelian diseases. In the conservation analysis the following species were considered: Human, Chimp, Gorilla, Rhesus, Tarsier, Mouse lemur, Bushbaby, Tree shrew, Mouse, Squirrel, Rabbit, Cow, Horse, Cat, Dog, Hedgehog, Elephant, Sloth, Wallaby, Opossum, Platypus, Chicken, Lizard, X. tropicalis, Tetraodon, Stickleback and Zebrafish. The conservation criteria have been described in the Suppl. Methods.

  based on cDNA sequence from the Ref. NM_201253.2, where A of the ATG initiation codon is 1. Lam AG -Laminin AG like domain; TMtransmembrane; Ccytoplasmic. LCA -Leber congenital amaurosis; RPretinitis pigmentosa; EORDearly onset retinal dystrophy; PPRPEpreservation of para-arteriolar retinal pigment epithelium. For PolyPhen-2 the HumVar value was taken, which is preferred for the diagnostic of human Mendelian diseases. In the conservation analysis the following species were considered: Human, Chimp, Gorilla, Rhesus, Tarsier, Mouse lemur, Bushbaby, Tree shrew, Mouse, Squirrel, Rabbit, Cow, Horse, Cat, Dog, Hedgehog, Elephant, Sloth, Wallaby, Opossum, Platypus, Chicken, Lizard, X. tropicalis, Tetraodon, Stickleback and Zebrafish. The conservation criteria have been described in the Suppl.

  numbering is based on cDNA sequence from the Ref. NM_201253.2, where A of the ATG initiation codon is 1. Lam AG -Laminin AG like domain; TMtransmembrane; Ccytoplasmic. For PolyPhen-2 the HumVar value was taken, which is preferred for the diagnostic of human Mendelian diseases. In the conservation analysis the following species were considered: Human, Chimp, Gorilla, Rhesus, Tarsier, Mouse lemur, Bushbaby, Tree shrew, Mouse, Squirrel, Rabbit, Cow, Horse, Cat, Dog, Hedgehog, Elephant, Sloth, Wallaby, Opossum, Platypus, Chicken, Lizard, X. tropicalis, Tetraodon, Stickleback and Zebrafish. The conservation criteria have been described in the Suppl. Methods.

Table 1 . Patients with CRB1 mutations identified in this study

 1

		Allele 1
	Patient number	Family

Table 2 : Clinical data

 2

	Patient	Age at time of testing	Age at time of diagnosis	Sex	Relevant medical and ophthalmology history	Family history	Symptoms	BCVA OD/OS Refraction	Lens	Fundus examination	OCT	FAF
	53	27	20	M	none	From Ivory Coast, 10 brothers and sisters, 1 sister affected	Night blindness at 6 then photophobia then decreased vision	LP 20/500 +2(-1.50)60° +1.75(-1.5)125°	Clear	Widespread vessels clumped pigment migration with no pale optic disc or narrowed retinal	Macular thinning with loss of ONL	Loss of AF at the posterior pole and periphery
	54	41	25	F	none	From French descent One affected brother	Night blindness	20/640 20/100 Prior to lens surgery: +5.50(-1)5° +5.50(-1)165°	IOL	Peripheral RPE changes with bone spicules, perifoveal atrophy, pale optic disc, narrowing of retinal vessels	Thinning of the ONL within the macular region	Loss of AF in the perifoveal region and outside the vascular arcades
										Peripheral RPE		Patchy loss of
	229	29	20	F	none	From French descent	Night blindness	20/80 20/50 +2(-0.75)5° +2.50(-1.50)5°	Clear	changes, little bone spicules, no pale optic disc or narrowed retinal	CME, thinning of ONL	AF in the periphery; foveal modification of AF due to the
										vessels, CME		CME
	368	13	12	F	Seizure in infancy	From Turkish descent maternal grand-mother said to be blind	photophobia	20/80 20/63 +6.50(-1.25)160° +6.50(-1)7°	Clear	Peripheral RPE changes with bone spicules, perifoveal atrophy, pale optic disc, narrowing of retinal vessels, CME	CME with relative preservation of foveal architecture	Patchy loss of AF outside the vascular arcades, foveal AF changes due to CME
	409	43	Teenage years	F	none	From Italian descent	Night blindness then photophobia	20/160 20/100 Plano Plano	Clear	Peripheral bone spicules with perifoveal atrophy	Thinning of the ONL	Loss of AF outside the vascular arcades and in the perifoveal area

(right eye); OS: Oculis Sinistra (left eye); IOL: intra ocular lens; CF: counting fingers; HM: hand motion; LP: light perception; RPE: retinal pigment epithelium; RP: retinitis pigmentosa; OHT: ocular hypertension; ONL: Outer Nuclear Layer

Table 3 : Function data

 3

	Patient	Colour vision (15 saturated Hue)	Binocular Goldman visual field, III4 isopter	Full field ERG	Multifocal ERG
	53	NP	Inf to 5°	ND	ND
	54	Dyschromatopsia without axis	Inf to 5°	ND	ND
	229	Normal	40 central degree with 2 peripheral island of perception	ND	ND
	368	Normal	120° horizontally, 60° vertically with relative central annular scotoma	Residual responses consistent with severe rod-cone dysfunction	Residual responses to central hexagones
	409	Dyschromatopsia without axis	100° horizontally, 60° vertically with annular scotoma	Residual cone responses	ND
	547	Bilateral tritaonopia	20 central degrees both horizontally and vertically	ND	ND
	1183	NP	Inf to 5°	ND	ND
	1731	OD NP, OS tritaonopia	5 central degrees	ND	ND
	03144	Normal	20 central degrees both horizontally and vertically	ND	ND
	3969	Dyschromatopsia without axis	20 central degree with 2 peripheral island of perception	ND	ND
	4240	Normal	130° vertically and 110° horizontally	30% decreased scotopic responses with photopic responses at the lower limit of normal	Decreased responses to central hexagones
	NP: not performed; ND: not detectable

Table 4 . Average prevalence of CRB1 mutations in retinal dystrophy patients in published reports

 4

	Dystrophy	Prevalence*	Patients with two CRB1 alleles	Patients with one CRB1 allele	Added cohort size	References
	LCA/EORD	10.1%	109	57	1645

because it co- segregates in the family (not found in 362 alleles)

										)
	c.2365_2367del	p.Asn789del	LamAG	Not conserved	-	-	1	EORD	this inframe	This study
	AAT, in frame		2	(considering 24					deletion is
	deletion			species, Ser in					likely
				Tarsier)					pathogenic,
	c.2401A>T	p.Lys801*	LamAG 2	protein	-	-	27	LCA, RP,
				truncation, NMD				EORD,
								PPRPE, ret
								talangiectas
								ia

Supplement Table S2. Unclassified nonsynonymous changes Exon Nucleotide change Aminoacid change Protein domain Effect/residue conservation SIFT predictions PolyPhen -2 predictions No. of reported alleles

		c.614T>C p.Ile205Thr c.2809G> p.Ala937Th	EGF5 EGF14	Moderately Highly	Tolerated Tolerated	Possibly Possibly	5 1	LCA, LCA	For this variant the second Considered as	(Bernal, et (Seong, et
		A	r		conserved in conserved in	(score 0.45) (score 0.13)	Damaging Damaging		EORD, RP	mutant CRB1 allele has polymorphism by the	al., 2003; al., 2008)
					vertebrates placental		(score 0.629) (score 0.838)			never been shown. It has authors, however it was not	den
					considering 26 mammals					been suggested as non-present in 170 controls, no	Hollander, et
					species (Val in (considering 16					pathogenic (den Holl2004). cosegregation data was	al., 2004;
					Mouse Lemur, species)					Cosegregation of this available. Due to high	Henderson,
					Opossum and					change has been shown with conservation and Polyphen2	et al., 2010;
					Stickleback)					a mutant allele from another prediction it is considered as	Vallespin, et
										parent (Vallespin 2007). unclassified variant	al., 2007b)
	9	c.3103C>	p.His1035T	LamA	Moderately	Tolerated	Benign	1	LCA/RP?	Digenic inheritence with Unknown second allele, not	(Henderson,
		T	yr	G 3	conserved	(score 1.00)	(score 0.027)			GUCY2D and RPGRIP1 found in 100 controls, no	et al., 2010)
					(considering 25					have been suggested in cosegregation information
					species, Tyr in					Villespin et al but the
					Cow)					digenic mutations did not
	11 alt c.4082G>	p.Arg1361	TM	Moderately	this	Benign	1	LCA	co-segregate. Unknown second allele;	(Simonelli,
		A	His		conserved (in	alternative	(score 0.010)			mutation in the alternative	et al., 2007)
					this case,	transcript			Phenotype	remarks transcript AF154671	reference
					conservation of	failed to be
	6	c.1472A>	p.Asp491V	LamA	Not conserved the Arg codon	Tolerated analysed	Benign	1	EORD	Considered as unclassified	(Coppieters,
	2 6 12	c.619G>A p.Val162M et T al c.1903T>C p.Ser635Pr o c.4060G> p.Ala1354T A hr	EGF4 G 1 LamA G 1 TM	Weakly conserved (considering 27 species; Met in (CGT) was considered in 23 species; in Hedgehog and Stickleback the Mouse, Rabbit, CAC codes for Cow, Dog) Weakly conserved (considering 27 species; Pro in His) Moderately conserved up to X. tropicalis (considering 22 Mouse lemour) species, Val in	Tolerated (score 0.25) (score 0.28) Tolerated (score 0.17)	Benign (score 0.023) (score 0.090) Benign (score 0.047)	1 1	PPCRA LCA	Dominant inheritance, not present in 150 controls, co-variant by the authors, p.Cys948Tyr was present on the second allele, no segregates in the family, cosegregation information LOD score: 1.8 Second mutation is a likeley pathogenic splice mutation, however no cosegregation analysis was performed	et al., 2010) (McKay, et al., 2005) (Li, et al., 2011)
					Mouse

Acknowledgments

The authors would like to thank patients and families for participation in this study, Dominique Santiard-Baron, Christine Chaumeil and clinical staff for their help in clinical data and DNA collection, Sandro Banfi, Robert Henderson and Qingjiong Zhang for additional information on genotype-phenotype correlations of previously published mutations and Robert Gillan for help with the manuscript. The project was financially supported by the Foundation Fighting Blindness (I.A. FFB Grant No: CD-CL-0808-0466-CHNO and the CIC503 recognized as an FFB center, FFB Grant No: C-CMM-0907-0428-INSERM04), Agence Nationale de la Recherche (SSB), Fondation Voir et Entendre (CZ), GIS-maladies rares (CZ), Ville de Paris and Région Ile de France, National Institutes of Health (USA) (KB NIH, Grant No: 1R01EY020902 -01A1).

Financial Support: Foundation Fighting Blindness (I.A. FFB Grant No: CD-CL-0808-0466-CHNO and the CIC503 recognized as an FFB center, FFB Grant No: C-CMM-0907-0428-INSERM04), Agence Nationale de la Recherche (SSB), Fondation Voir et Entendre (CZ), GIS-maladies rares (CZ), Ville de Paris and Région Ile de France, National Institutes of Health (USA) (KB NIH, Grant No: 1R01EY020902 -01A1).

Supplement Table S1. Likely pathogenic mutations in CRB1

 (Bernal, et al., 2003;Vallespin, et al., 2007b) [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]Henderson, et al., 2010;Vallespin, et al., 2007b;Yzer, et al., 2006) (Bernal, et al., 2003;Booij, et al., 2005;Clark, et al., 2010;[START_REF] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes[END_REF][START_REF] Hollander | CRB1 mutation spectrum in inherited retinal dystrophies[END_REF]den Hollander, et al., 2001a;[START_REF] Hollander | Identification of novel mutations in patients with Leber congenital amaurosis and juvenile RP by genome-wide homozygosity mapping with SNP microarrays[END_REF][START_REF] Hollander | Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12)[END_REF]Galvin, et al., 2005;[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]Henderson, et al., 2010;Henderson, et al., 2007;Jacobson, et al., 2003;Lotery, et al., 2001a;[START_REF] Riveiro-Alvarez | Molecular analysis of ABCA4 and CRB1 genes in a Spanish family segregating both Stargardt disease and autosomal recessive retinitis pigmentosa[END_REF][START_REF] Tosi | Case report: autofluorescence imaging and phenotypic variance in a sibling pair with early-onset retinal dystrophy due to defective CRB1 function[END_REF]Vallespin, et al., 2007b;Yzer, et al., 2006;[START_REF] Zernant | Genotyping microarray (disease chip) for Leber congenital amaurosis: detection of modifier alleles[END_REF]This [START_REF] Benayoun | Genetic heterogeneity in two consanguineous families segregating early onset retinal degeneration: the pitfalls of homozygosity mapping[END_REF][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]Simonelli, et al., 2007) [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]Lotery, et al., 2001a [START_REF] Benayoun | Genetic heterogeneity in two consanguineous families segregating early onset retinal degeneration: the pitfalls of homozygosity mapping[END_REF][START_REF] Gerber | A novel mutation disrupting the cytoplasmic domain of CRB1 in a large consanguineous family of Palestinian origin affected with Leber congenital amaurosis[END_REF][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] Coppieters | Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes[END_REF]den Hollander, et al., 2001b;[START_REF] Hollander | Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12)[END_REF]Yzer, et al., 2006