Basal exon skipping and nonsense-associated altered splicing allows bypassing complete CEP290 loss-of-function in individuals with unusually mild retinal disease

Basal exon skipping and nonsense-associated altered splicing allows bypassing complete CEP290... Abstract CEP290 mutations cause a spectrum of ciliopathies from Leber congenital amaurosis type 10 (LCA10) to embryo-lethal Meckel syndrome (MKS). Using panel-based molecular diagnosis testing for inherited retinal diseases, we identified two individuals with some preserved vision despite biallelism for presumably truncating CEP290 mutations. The first one carried a homozygous 1 base pair deletion in Exon 17, introducing a premature termination codon (PTC) in Exon 18 (c.1666del; p.Ile556Phefs*17). mRNA analysis revealed a basal exon skipping (BES) of Exon 18, providing mutant cells with the ability to escape protein truncation, while disrupting the reading frame in controls. The second individual harbored compound heterozygous nonsense mutations in Exon 8 (c.508A>T, p.Lys170*) and Exon 32 (c.4090G>T, p.Glu1364*), respectively. Some CEP290 lacking Exon 8 were detected in mutant fibroblasts but not in controls whereas some skipping of Exon 32 occurred in both lines, but with higher amplitude in the mutant. Considering that the deletion of either exon maintains the reading frame in either line, skipping in mutant cells likely involves nonsense-associated altered splicing alone (Exon 8), or with BES (Exon 32). Skipping of PTC-containing exons in mutant cells allowed production of CEP290 isoforms with preserved ability to assemble into a high molecular weight complex and to interact efficiently with proteins important for cilia formation and intraflagellar trafficking. In contrast, studying LCA10 and MKS fibroblasts we show moderate to severe cilia alterations, providing support for a correlation between disease severity and the ability of cells to express shortened, yet functional, CEP290 isoforms. Introduction Ciliopathies are a large group of Mendelian disorders caused by direct or indirect dysfunction of primary or motile cilia found on most vertebrate cells (1). In humans, ciliopathies are characterized by prominent genetic heterogeneity and pleiotropy. Mutations in CEP290 (MIM610142, https://www.omim.org; date last accessed May 22, 2018), encoding the 290-kDa centrosomal protein, have been involved in a large spectrum of ciliopathies, ranging from isolated retinal dystrophy to multi-visceral and sometimes embryo-lethal Meckel syndrome type 4 (MKS; MIM611134) (2). It has been proposed that the origin of CEP290 pleiotropy lies not in the effects of mutations on protein function, but rather in the effects of mutations on the amount of protein retaining all or some of the full-length CEP290 functionality, which can be supplied to the cell (3). Consistent with this view, isolated retinal disease has been ascribed to hypomorphic mutations, the most frequent of which is a deep intronic change c.2991+1655A>G, which introduces a cryptic exon and a premature termination codon (PTC) in 50–75% of the CEP290 mRNA products (4,5). Other hypomorphic variants have been reported to preferentially cluster in exons, which experience spontaneous non-canonical skipping through a mechanism referred to as CEP290 basal exon skipping (BES), which likely enables the production of low levels of near-full length functional protein from mutation-free mRNA with intact reading frames (3). The CEP290-retinal disease typically presents as a neonatal-onset and dramatically severe cone-rod dystrophy, known as Leber congenital amaurosis (LCA) type I (6,7) (MIM24000). The report of selective exclusion of a CEP290 exon with a stop codon in individuals with unexpectedly mild retinal disease (8,9) has suggested that nonsense-associated altered splicing (NAS) may be another mechanism and, in the light of the attenuated phenotype, likely a more efficient one, that provides cells the potential to compensate for deleterious mutations. However, no analysis supported this hypothesis. Using gene panel-based sequencing in a series of individuals with retinal dystrophies (RD) of variable presentation, we found homozygosity for a one base pair (bp) deletion and compound heterozygosity for nonsense mutations in two unrelated adult individuals with unusually preserved central vision. Here, we report that, despite biallelism for apparent truncating mutations, fibroblasts from the patients produced minimally shortened CEP290 proteins, which retained functional interactions important to cilia formation and intraflagellar trafficking. We show that BES and/or NAS of exons allowed bypassing PTC in the cells from the two individuals. Results Panel-based molecular diagnosis testing identifies biallelism for presumably truncating CEP290 mutations in two individuals (P1 and P2) affected with an unusually moderate retinal disease Analyzing a molecular diagnosis panel of 190 genes for inherited retinal diseases in a large series individuals affected with RD of variable age, mode of onset and clinical presentation (article in preparation), we identified homozygosity and compound heterozygosity for presumably truncating CEP290 mutations in two unrelated individuals (P1 and P2): a 1 bp deletion in Exon 17 predicted to introduce a PTC in Exon 18 and nonsense mutations in Exon 8 (c.508A>T, p.Lys170*) and Exon 32 (c.4090G>T, p.Glu1364*), respectively (Fig. 1). In contrast with CEP290 genotype–phenotype correlations (4,7), the two individuals had notable preservation of central vision over two to three decades despite early-onset and severe retinal dystrophy (Table 1). Table 1. Mutations and clinical features of individuals included in the study Name Agea (year) CEP290 mutations Phenotype References Control-1; C1 8 None No overt pathology – Control-2; C2 10 None No overt pathology – Patient II: 2 (family 1) P1 18 c.1666del (p.Ile556Phefs*17) c.1666del (p.Ile556Phefs*17) EOSRD No nystagmus, no photophobia Photo-attraction, night discomfort Scotopic and photopic ERG non-recordable Visual acuity: RE=20/40, LE=20/30 – Patient II: 1 (family 2) P2 33 c.508A>T (p.Lys170*) c.4090G>T (p.Glu1364*) EOSRD No nystagmus, no photophobia Photo-attraction, night discomfort Scotopic and photopic ERG non-recordable Visual acuity: RE=20/100, LE=20/400 – LCA10_1 3 c.2991 + 1655A>G (p.Cys998*) c.2991 + 1655A>G (p.Cys998*) LCA Nystagmus, photophobia Scotopic and photopic ERG non-recordable No visual acuity (5) LCA10_2 33 c.2991 + 1655A>G (p.Cys998*) c.2991 + 1655A>G (p.Cys998*) LCA Nystagmus, photophobia Scotopic and photopic ERG non-recordable No visual acuity (5) MKS Embryo c.613C>T (p.Arg205*) c.613C>T (p.Arg205*) MKS (24) Name Agea (year) CEP290 mutations Phenotype References Control-1; C1 8 None No overt pathology – Control-2; C2 10 None No overt pathology – Patient II: 2 (family 1) P1 18 c.1666del (p.Ile556Phefs*17) c.1666del (p.Ile556Phefs*17) EOSRD No nystagmus, no photophobia Photo-attraction, night discomfort Scotopic and photopic ERG non-recordable Visual acuity: RE=20/40, LE=20/30 – Patient II: 1 (family 2) P2 33 c.508A>T (p.Lys170*) c.4090G>T (p.Glu1364*) EOSRD No nystagmus, no photophobia Photo-attraction, night discomfort Scotopic and photopic ERG non-recordable Visual acuity: RE=20/100, LE=20/400 – LCA10_1 3 c.2991 + 1655A>G (p.Cys998*) c.2991 + 1655A>G (p.Cys998*) LCA Nystagmus, photophobia Scotopic and photopic ERG non-recordable No visual acuity (5) LCA10_2 33 c.2991 + 1655A>G (p.Cys998*) c.2991 + 1655A>G (p.Cys998*) LCA Nystagmus, photophobia Scotopic and photopic ERG non-recordable No visual acuity (5) MKS Embryo c.613C>T (p.Arg205*) c.613C>T (p.Arg205*) MKS (24) a Age at which the dermal biopsy and the last clinical examination were performed. EOSRD, early onset and severe retinal dystrophy; LCA, Leber congenital amaurosis; MKS, Meckel syndrome; ERG, electroretinogram. Table 1. Mutations and clinical features of individuals included in the study Name Agea (year) CEP290 mutations Phenotype References Control-1; C1 8 None No overt pathology – Control-2; C2 10 None No overt pathology – Patient II: 2 (family 1) P1 18 c.1666del (p.Ile556Phefs*17) c.1666del (p.Ile556Phefs*17) EOSRD No nystagmus, no photophobia Photo-attraction, night discomfort Scotopic and photopic ERG non-recordable Visual acuity: RE=20/40, LE=20/30 – Patient II: 1 (family 2) P2 33 c.508A>T (p.Lys170*) c.4090G>T (p.Glu1364*) EOSRD No nystagmus, no photophobia Photo-attraction, night discomfort Scotopic and photopic ERG non-recordable Visual acuity: RE=20/100, LE=20/400 – LCA10_1 3 c.2991 + 1655A>G (p.Cys998*) c.2991 + 1655A>G (p.Cys998*) LCA Nystagmus, photophobia Scotopic and photopic ERG non-recordable No visual acuity (5) LCA10_2 33 c.2991 + 1655A>G (p.Cys998*) c.2991 + 1655A>G (p.Cys998*) LCA Nystagmus, photophobia Scotopic and photopic ERG non-recordable No visual acuity (5) MKS Embryo c.613C>T (p.Arg205*) c.613C>T (p.Arg205*) MKS (24) Name Agea (year) CEP290 mutations Phenotype References Control-1; C1 8 None No overt pathology – Control-2; C2 10 None No overt pathology – Patient II: 2 (family 1) P1 18 c.1666del (p.Ile556Phefs*17) c.1666del (p.Ile556Phefs*17) EOSRD No nystagmus, no photophobia Photo-attraction, night discomfort Scotopic and photopic ERG non-recordable Visual acuity: RE=20/40, LE=20/30 – Patient II: 1 (family 2) P2 33 c.508A>T (p.Lys170*) c.4090G>T (p.Glu1364*) EOSRD No nystagmus, no photophobia Photo-attraction, night discomfort Scotopic and photopic ERG non-recordable Visual acuity: RE=20/100, LE=20/400 – LCA10_1 3 c.2991 + 1655A>G (p.Cys998*) c.2991 + 1655A>G (p.Cys998*) LCA Nystagmus, photophobia Scotopic and photopic ERG non-recordable No visual acuity (5) LCA10_2 33 c.2991 + 1655A>G (p.Cys998*) c.2991 + 1655A>G (p.Cys998*) LCA Nystagmus, photophobia Scotopic and photopic ERG non-recordable No visual acuity (5) MKS Embryo c.613C>T (p.Arg205*) c.613C>T (p.Arg205*) MKS (24) a Age at which the dermal biopsy and the last clinical examination were performed. EOSRD, early onset and severe retinal dystrophy; LCA, Leber congenital amaurosis; MKS, Meckel syndrome; ERG, electroretinogram. Figure 1. View largeDownload slide Identification of CEP290 truncating mutations in two affected individuals with unusually preserved central vision. (A, B) Pedigrees, segregation analysis and Sanger sequencing chromatograms of DNA showing the CEP290 mutations in Families 1 and 2. Filled and unfilled symbols represent affected and unaffected members, respectively. The affected individual P1 (II: 2, Family 1) is homozygous for the c.1666del (p.Ile556Phefs*17) mutation, whereas the affected individual P2 (II: 1, Family 2) is compound heterozygous for the c.508A>T (p.Lys170*) and c.4090G>T (p.Glu1364*) mutations. The biparental transmission is confirmed by the detection of mutations in single heterozygosity in parental DNAs. Red arrows indicate mutant nucleotide positions. Symbol+represents the WT allele. Figure 1. View largeDownload slide Identification of CEP290 truncating mutations in two affected individuals with unusually preserved central vision. (A, B) Pedigrees, segregation analysis and Sanger sequencing chromatograms of DNA showing the CEP290 mutations in Families 1 and 2. Filled and unfilled symbols represent affected and unaffected members, respectively. The affected individual P1 (II: 2, Family 1) is homozygous for the c.1666del (p.Ile556Phefs*17) mutation, whereas the affected individual P2 (II: 1, Family 2) is compound heterozygous for the c.508A>T (p.Lys170*) and c.4090G>T (p.Glu1364*) mutations. The biparental transmission is confirmed by the detection of mutations in single heterozygosity in parental DNAs. Red arrows indicate mutant nucleotide positions. Symbol+represents the WT allele. In silico analysis suggests that CEP290 mutations carried by P1 and P2 affect splicing The apparent inconsistency between phenotypes and genotypes in P1 and P2 individuals led us to analyze the effect of their mutations on splicing. The use of prediction software solutions analyzing splice signals and exonic splicing silencer (ESS)/exonic splicing enhancer (ESE) binding sites, suggests that the c.1666del identified in P1 in homozygosity does not modify significant consensus splice site scores. However, it decreases Exon 17 ESS/ESE ratio, thus reducing the susceptibility of skipping compared with the wild-type (WT) sequence (Table 2). The c.508A>T and c.4090G>T changes identified in compound heterozygosity in P2 do not modify the consensus splice site strength either. However, in contrast to the c.1666del, they increased the ESS/ESE ratios, conferring mutant Exons 8 and 32 with a higher chance of skipping than WT counterparts (Table 2). Additionally, the c.4090G>T allele is expected to create a strong donor splice-site 60 bp downstream of the start of Exon 32 (Supplementary Material, Fig. S1). Table 2. Impact of mutations on ESS and ESE motifs EX-SKIP predictions HOT-SKIP predictions Nucleotide ESS ESE ESS/ESE ESS ESE ESS/ESE Skipping predictions of mutant allele compared with WT allele c.508 Exon 8 A 0 21 0 0 10 0 – T 1 13 0.08 1 2 0.5 Higher chance G 0 20 0 0 9 0 Comparable chance C 0 24 0 0 13 0 Comparable chance c.1666 exon17 A 16 97 0.16 N.A. – delA 14 99 0.14 Lower chance c.4090 Exon 32 A 9 66 0.14 1 7 0.14 Lower chance T 12 60 0.20 4 1 4 Higher chance G 10 64 0.16 2 5 0.4 – C 8 59 0.14 0 0 0 Lower chance EX-SKIP predictions HOT-SKIP predictions Nucleotide ESS ESE ESS/ESE ESS ESE ESS/ESE Skipping predictions of mutant allele compared with WT allele c.508 Exon 8 A 0 21 0 0 10 0 – T 1 13 0.08 1 2 0.5 Higher chance G 0 20 0 0 9 0 Comparable chance C 0 24 0 0 13 0 Comparable chance c.1666 exon17 A 16 97 0.16 N.A. – delA 14 99 0.14 Lower chance c.4090 Exon 32 A 9 66 0.14 1 7 0.14 Lower chance T 12 60 0.20 4 1 4 Higher chance G 10 64 0.16 2 5 0.4 – C 8 59 0.14 0 0 0 Lower chance Effect of nucleotide changes at positions c.508, c.1666 and c.4090 on ESS and ESE motifs according to EX-SKIP and HOT-SKIP prediction programs. The WT and mutant alleles identified in this study are labeled in blue and red, respectively. N.A., not applicable; ESS, exonic splicing silencer; ESE, exonic splicing enhancer. Table 2. Impact of mutations on ESS and ESE motifs EX-SKIP predictions HOT-SKIP predictions Nucleotide ESS ESE ESS/ESE ESS ESE ESS/ESE Skipping predictions of mutant allele compared with WT allele c.508 Exon 8 A 0 21 0 0 10 0 – T 1 13 0.08 1 2 0.5 Higher chance G 0 20 0 0 9 0 Comparable chance C 0 24 0 0 13 0 Comparable chance c.1666 exon17 A 16 97 0.16 N.A. – delA 14 99 0.14 Lower chance c.4090 Exon 32 A 9 66 0.14 1 7 0.14 Lower chance T 12 60 0.20 4 1 4 Higher chance G 10 64 0.16 2 5 0.4 – C 8 59 0.14 0 0 0 Lower chance EX-SKIP predictions HOT-SKIP predictions Nucleotide ESS ESE ESS/ESE ESS ESE ESS/ESE Skipping predictions of mutant allele compared with WT allele c.508 Exon 8 A 0 21 0 0 10 0 – T 1 13 0.08 1 2 0.5 Higher chance G 0 20 0 0 9 0 Comparable chance C 0 24 0 0 13 0 Comparable chance c.1666 exon17 A 16 97 0.16 N.A. – delA 14 99 0.14 Lower chance c.4090 Exon 32 A 9 66 0.14 1 7 0.14 Lower chance T 12 60 0.20 4 1 4 Higher chance G 10 64 0.16 2 5 0.4 – C 8 59 0.14 0 0 0 Lower chance Effect of nucleotide changes at positions c.508, c.1666 and c.4090 on ESS and ESE motifs according to EX-SKIP and HOT-SKIP prediction programs. The WT and mutant alleles identified in this study are labeled in blue and red, respectively. N.A., not applicable; ESS, exonic splicing silencer; ESE, exonic splicing enhancer. RT-PCR analysis shows selective exclusion of PTC-containing exons in fibroblasts from P1 and P2 individuals Analysis of CEP290 mRNA from the patient and control skin-fibroblasts were consistent with the in silico predictions. Agarose gel analysis and Sanger sequencing of RT-PCR products generated from P1 mRNA using primers specific to Exons 15 and 19 (Supplementary Material, Fig. S2 and Table S1) detected a full-length isoform, which carried the c.1666del in Exon 17, and a shorter mRNA lacking Exon 18 (skipped isoform) (Fig. 2A andSupplementary Material, Fig. S3A). The c.1666del in the full-length mutant mRNA is predicted to disrupt the reading frame and introduce a PTC in Exon 18 (p.Ile556Phefs*17). Interestingly, in the skipped mRNA, the deletion of Exon 18 in combination with the 1 bp deletion in Exon 17 allows for maintaining an intact reading frame (Fig. 2A). In control fibroblasts, very low levels of CEP290 cDNA lacking Exon 18 could be detected, suggesting that this exon undergoes BES (Fig. 2A). Unlike the mutant, WT Exon 17 is not in frame with Exon 19 (Fig. 3). Thus, the skipping of Exon 18 introduces a PTC. Figure 2. View largeDownload slide Naturally occurring exclusion of CEP290 exons encompassing premature stop codon. (A–C) Analysis of reverse transcribed CEP290 mRNA extracted from human fetal retina (Retina), patient (P1 and P2) and control (C1) fibroblasts. Images of agarose gel showing RT-PCR fragments produced using primer pairs surrounding mutant Exons 17, 8 and 32, respectively. White asterisks point to heteroduplex products. The boxes summarize the exonic organization and phasing of each RT-PCR product. Red arrows show the position of the PTC within CEP290 isoforms. Numbers next to boxes refer to corresponding Sanger sequencing chromatograms represented in Supplementary Material, Figure S3. (D–F) Relative expression of WT (gray bars) and mutant (black bars) full-length isoforms, and (G–I) skipped (CEP290Δ18, CEP290Δ8 and CEP290Δ32; hatched bars) CEP290 mRNAs in human fetal retina (Retina), control (C1 and C2) and patient (P1 and P2) fibroblasts as determined by RT-qPCR using GUSB and RPLP0 genes as reference. C corresponds to C1 and C2 pooled values. Values are the mean±SEM derived from three independent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n.s., not significant. Figure 2. View largeDownload slide Naturally occurring exclusion of CEP290 exons encompassing premature stop codon. (A–C) Analysis of reverse transcribed CEP290 mRNA extracted from human fetal retina (Retina), patient (P1 and P2) and control (C1) fibroblasts. Images of agarose gel showing RT-PCR fragments produced using primer pairs surrounding mutant Exons 17, 8 and 32, respectively. White asterisks point to heteroduplex products. The boxes summarize the exonic organization and phasing of each RT-PCR product. Red arrows show the position of the PTC within CEP290 isoforms. Numbers next to boxes refer to corresponding Sanger sequencing chromatograms represented in Supplementary Material, Figure S3. (D–F) Relative expression of WT (gray bars) and mutant (black bars) full-length isoforms, and (G–I) skipped (CEP290Δ18, CEP290Δ8 and CEP290Δ32; hatched bars) CEP290 mRNAs in human fetal retina (Retina), control (C1 and C2) and patient (P1 and P2) fibroblasts as determined by RT-qPCR using GUSB and RPLP0 genes as reference. C corresponds to C1 and C2 pooled values. Values are the mean±SEM derived from three independent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n.s., not significant. Figure 3. View largeDownload slide Human CEP290 exon map and phasing. Schematic representation of the 54 exons of the human CEP290 gene. The black box corresponds to non-coding Exon 1. The start codon (ATG) in Exon 2 is represented by an arrow and the stop codon (TAA) is indicated at the end of Exon 54. Light gray boxes represent exons for which skipping would not disrupt the reading frame. Dark gray boxes indicate exons for which skipping would disrupt the reading frame. Protein interaction domains are represented above and/or below the sequence [based on references (26,27)]. Figure 3. View largeDownload slide Human CEP290 exon map and phasing. Schematic representation of the 54 exons of the human CEP290 gene. The black box corresponds to non-coding Exon 1. The start codon (ATG) in Exon 2 is represented by an arrow and the stop codon (TAA) is indicated at the end of Exon 54. Light gray boxes represent exons for which skipping would not disrupt the reading frame. Dark gray boxes indicate exons for which skipping would disrupt the reading frame. Protein interaction domains are represented above and/or below the sequence [based on references (26,27)]. Considering P2 fibroblasts, RT-PCR using primers specific to Exons 6 and 10, and 31 and 34 (Supplementary Material, Fig. S2 and Table S1), generated three and four PCR products, the sequencing of which identified (Supplementary Material, Fig. S3B and C): mutant (c.508A) and WT (c.508T) sequences encompassing Exons 6–10, an isoform lacking Exon 8, mutant (c.4090T) and WT (c.4090G) sequences encompassing Exons 31–34, an isoform lacking Exon 32 and another lacking the last 106 nucleotides of Exon 32, respectively (Fig. 2B and C). The complete deletion of Exon 8 or 32 removes the PTC, while maintaining the reading frame (Fig. 3). In contrast, the partial deletion of Exon 32 removes the c.4090G>T nonsense mutation yet disrupts the reading frame and introducing another PTC (p.Glu1364Phefs*20; Fig. 2C;Supplementary Material, Fig. S3C). In the controls, we detected the full-length CEP290 mRNA, but not the isoform deleted of Exon 8 (Fig. 2B). Considering that the skipping of WT Exon 8 would produce an in-frame NMD-resistant isoform, this observation suggests that this exon does not undergo BES in fibroblasts and that CEP290 lacking Exon 8 in P2 cells arose exclusively from the mutant allele. In contrast, we observed some Exon 32 skipping in the controls, yet with a significantly lower amplitude than in P2 cells. Because the deletion of this exon does not modify the reading frame in either cell lines, this observation suggests that both BES and NAS occurred in P2 cells (Fig. 2B and C). Analysis of mRNA from human retina supports BES of Exon 18 and 32 as in fibroblasts Consistent with what we observed in fibroblasts, agarose gel analysis and Sanger sequencing of RT-PCR products generated from human retina mRNA using primers flanking Exons 8, 18 and 32 (Supplementary Material, Fig. S2; Table S1) detected full-length CEP290 and mRNA isoforms lacking either Exon 18 or 32 but not Exon 8 (Fig. 2A–C;Supplementary Material, Fig. S3). RT-qPCR analysis supports NMD of PTC-encoding isoforms We determined the abundance of CEP290 in patient and control fibroblasts and in the retina by RT-qPCR using primers specific to full-length mRNAs and all skipped isoforms, except for the one partially deleted of Exon 32 (Supplementary Material, Fig. S2 and Table S2). This analysis revealed moderate amounts of CEP290 isoforms lacking either Exon 8, 18 or 32 in fibroblasts from P1 and P2 individuals (Fig. 2G–I). Since these isoforms have intact open reading frames free of PTC, this observation suggests that only reduced numbers of pre-mRNA copies underwent skipping in the fibroblasts from patients. In addition, we observed an under-representation of CEP290 isoforms encoding PTCs (full-length mutant mRNAs in P1 and P2 fibroblasts and mRNAs lacking Exon 18 in controls), compared with their counterparts, which have intact reading frames (full-length mRNAs in controls, skipped mRNAs in P1) (Fig. 2D–G). Consistent with the NMD of PTC-containing isoforms, their abundance significantly increased when fibroblasts were treated using an NMD inhibitor (emetine: 25 μg/ml, 15 h) (Supplementary Material, Fig. S4). In the retina, the full-length CEP290 mRNA was significantly more abundant than in control fibroblasts, supporting a higher CEP290 expression in the retina (4- to 5-fold) (Fig. 2D and F). Consistently, both the PTC-containing and PTC-free isoforms lacking Exons 18 and 32 were more abundant in the retina than in control fibroblasts (Fig. 2G and I). P1 and P2 fibroblasts express a CEP290 protein that localizes at the centrosome Protein lysates from 90 to 100% confluent mutant and control fibroblasts were analyzed by western blot using antibodies that recognize the carboxy-terminus extension of CEP290 and β-actin, respectively. We observed a band ∼290 kDa in P1 and P2 lysates as in controls, LCA10_1 and LCA10_2, and the abundance relative to β-actin of which varied strikingly (Fig. 4). In contrast, CEP290 was undetectable in MKS cells (Fig. 4A). Immunocytochemistry in 90–100% confluent serum-starved cells revealed CEP290 staining at the centrosome in P1 and P2 cells (Fig. 5A and B) with mean immunofluorescence intensities ∼30% of that of the controls (Fig. 5C). A comparable staining was observed in cells from LCA10_1 and LCA10_2 individuals (Fig. 5). These results are consistent with the view that, like WT CEP290, mRNAs deleted from Exons 18 and 8 and/or 32 produce minimally shortened, yet stable, CEP290 proteins, which have the ability to localize at the centrosome. Interestingly, although western blot analysis did not detect CEP290, immunocytochemistry disclosed some CEP290 positive MKS cells (Fig. 5). Figure 4. View largeDownload slide Effect of the selective exclusion of PTC-encoding exons on CEP290 protein production. (A) Immunodetection of the CEP290 protein in mutant fibroblasts (P1, P2, LCA10_1, LCA10_2 and MKS) and control cell lines (C1 and C2). β-Actin was used for normalization. (B) Quantification of CEP290 protein abundance. C corresponds to C1 and C2 pooled values. Values were determined by computed-densitometry analysis of CEP290 and β-actin expression in each sample and are the mean±SEM derived from five independent experiments. Whereas we measured a significant difference in CEP290 abundance between P1 and P2, there is none for LCA10_1 and LCA10_2 samples. *P<0.05, ****P<0.0001; n.s., not significant. Figure 4. View largeDownload slide Effect of the selective exclusion of PTC-encoding exons on CEP290 protein production. (A) Immunodetection of the CEP290 protein in mutant fibroblasts (P1, P2, LCA10_1, LCA10_2 and MKS) and control cell lines (C1 and C2). β-Actin was used for normalization. (B) Quantification of CEP290 protein abundance. C corresponds to C1 and C2 pooled values. Values were determined by computed-densitometry analysis of CEP290 and β-actin expression in each sample and are the mean±SEM derived from five independent experiments. Whereas we measured a significant difference in CEP290 abundance between P1 and P2, there is none for LCA10_1 and LCA10_2 samples. *P<0.05, ****P<0.0001; n.s., not significant. Figure 5. View largeDownload slide CEP290 expression in quiescent cells. (A) Representative images of CEP290 (green) localization in control and mutant fibroblasts induced to quiescence. Acetylated α-tubulin (Ac-tub; red) is used to mark the ciliary axoneme. As in control cell lines (C1 and C2), CEP290 is correctly localized at the base of the cilia in patient (P1 and P2) and LCA10 (LCA10_1 and LCA10_2) fibroblasts. CEP290 in MKS cells was almost undetectable. Scale bar, 5 µm. (B) Centrosomal localization of CEP290 (green) in control and mutant fibroblasts. The gamma-tubulin (γ-tub; red) labeling is used as a centrosomal marker. Image scale bar, 5 µm. Inset scale bar, 2 µm. (C) Quantification of the CEP290 immunofluorescence intensity at the basal body in each cell line. Values are the mean±SEM. Immunolabeling was performed from 90 to 100% confluent cells in two independent experiments. Automatic intensity measures were recorded in four to five fields. C corresponds to C1 and C2 pooled values. ****P<0.0001; n.s., not significant; A.U., arbitrary unit. Figure 5. View largeDownload slide CEP290 expression in quiescent cells. (A) Representative images of CEP290 (green) localization in control and mutant fibroblasts induced to quiescence. Acetylated α-tubulin (Ac-tub; red) is used to mark the ciliary axoneme. As in control cell lines (C1 and C2), CEP290 is correctly localized at the base of the cilia in patient (P1 and P2) and LCA10 (LCA10_1 and LCA10_2) fibroblasts. CEP290 in MKS cells was almost undetectable. Scale bar, 5 µm. (B) Centrosomal localization of CEP290 (green) in control and mutant fibroblasts. The gamma-tubulin (γ-tub; red) labeling is used as a centrosomal marker. Image scale bar, 5 µm. Inset scale bar, 2 µm. (C) Quantification of the CEP290 immunofluorescence intensity at the basal body in each cell line. Values are the mean±SEM. Immunolabeling was performed from 90 to 100% confluent cells in two independent experiments. Automatic intensity measures were recorded in four to five fields. C corresponds to C1 and C2 pooled values. ****P<0.0001; n.s., not significant; A.U., arbitrary unit. The CEP290 protein produced in P1 and P2 cells are able to incorporate into a macromolecular complex CEP290 has been reported to contribute to a macromolecule as large as 2–3 MDa (10). To determine whether the CEP290 protein produced in P1 and P2 cells retained this ability, protein extracts were analyzed by BN-PAGE using antibodies specific to CEP290 and Vinculin (loading control). A purified mitochondrial membrane protein extract was loaded onto the gel to use 1 MDa mitochondrial complex 1 (revealed by an anti-GRIM19 antibody) as a high molecular weight protein marker. Consistent with western blot analysis, the CEP290 antibody revealed no complex in the MKS protein extract (Fig. 6). In all other cell lines, we observed a macromolecular complex exceeding 1 MDa (Fig. 6). We conclude that the loss of amino acid residues encoded by Exons 18 and 8 and/or 32 does not significantly impair the capability of CEP290 to interact with some or all its partners in the complex (Fig. 3). Figure 6. View largeDownload slide Assembly of CEP290 in a high-molecular weight protein complex. Blue native-polyacrylamide gel electrophoresis analysis of protein extracts from control (C1 and C2) and mutant (P1, P2, LCA10_1, LCA10_2 and MKS) fibroblasts. Immunodetection using a CEP290 antibody reveals the presence of a high molecular weight complex in all cells lines but the MKS. Vinculin complex detection serves as a loading control. OXPHOS proteins from isolated mitochondrial membranes (Mt extract) were loaded and complex 1 of the respiratory chain (Mt Complex I) was immunolabeled using an anti-GRIM19 antibody to serve as the high molecular weight (1 MDa) marker. Figure 6. View largeDownload slide Assembly of CEP290 in a high-molecular weight protein complex. Blue native-polyacrylamide gel electrophoresis analysis of protein extracts from control (C1 and C2) and mutant (P1, P2, LCA10_1, LCA10_2 and MKS) fibroblasts. Immunodetection using a CEP290 antibody reveals the presence of a high molecular weight complex in all cells lines but the MKS. Vinculin complex detection serves as a loading control. OXPHOS proteins from isolated mitochondrial membranes (Mt extract) were loaded and complex 1 of the respiratory chain (Mt Complex I) was immunolabeled using an anti-GRIM19 antibody to serve as the high molecular weight (1 MDa) marker. The functional network of interactions between CEP290, member RAS oncogene family, centrosomal protein of 110 kDa and pericentriolar material 1 and cilia formation is apparently unaltered in P1 and P2 fibroblasts CEP290 is localized at the centrosome through its interactions with pericentriolar material 1 (PCM1), member RAS oncogene family (RAB8A) and centrosomal protein of 110 kDa (CP110). During the transition of the cells from proliferation to quiescence, the primary cilia formation-suppressor CP110 is released. This leads to the disinhibition of CEP290 and RAB8A to promote ciliation and the relocalization of PCM1 in the pericentriolar zone (10–12). We examined the localization of these proteins by immunocytochemistry in quiescent fibroblasts. Consistent with the near-absence of CEP290 in the MKS cells, we observed no RAB8A signals in the few cells able to build a cilium (Fig. 7A and B), as well as PCM1 and CP110 concentric centrosomal accumulation (Fig. 7C–F). Ciliation was severely altered with very few cells having the ability to produce a cilium, the length of which was substantially abnormal (Fig. 8). In contrast, in P1 and P2 fibroblasts, similar to in LCA10_1 and LCA10_2 cells expressing some WT CEP290, RAB8A and CP110 signals were comparable to that in the controls (Fig. 7A–D). Considering PCM1, scatter dot plot representations show a compact distribution of signal intensities at centrosomes in P1 and P2 as in control cells. However, mean signal intensities were slightly increased in patient lines, suggesting a marginal accumulation at centrosomes (Fig. 7E and F). In contrast, in LCA10_1 and LCA10_2 cells (like in the MKS line though to a lesser extent), the distribution of PCM1 signal intensities was rather dispersed, with significantly higher mean signal intensities compared with the controls. This observation suggests that PCM1 accumulates at the centrosomes of most LCA10_1 and LCA10_2 cells (Fig. 7E and F). Interestingly, ciliation was apparently unaltered in P1 and P2, but not in LCA10_1 and LCA10_2, which have decreased cilia abundance and shorter axonemes (Fig. 8), suggesting a possible correlation between PCM1 redistribution and ciliation alterations. Figure 7. View largeDownload slide Localization and abundance of CEP290 centriolar satellite partners. (A) Representative images of RAB8A (red) localization in the cilia from control (C1 and C2) and mutant (P1, P2, LCA10_1, LCA10_2 and MKS) fibroblasts induced to quiescence. ARL13B (green) labeling is used to mark the ciliary membrane. Image scale bar, 5 µm. Inset scale bar, 5 µm. (B) Quantification of RAB8A-positive cilia. Values are the mean±SEM (n>80; two independent experiments). C corresponds to C1 and C2 pooled values. ***P<0.001, ****P<0.0001; n.s., not significant; A.U., Arbitrary unit. Representative images of (C) CP110 or (E) PCM1 (green) centrosomal staining in quiescent control and mutant fibroblasts. Centrosomes are labeled by gamma-tubulin (γ-tub.; red). Image scale bar, 5 µm. Inset scale bar, 2 µm. Quantification of (D) CP110 or (F) PCM1 immunofluorescence intensity at centrosomes in quiescent fibroblasts. Values are the mean±SEM. Both immunolabelings were performed from 90 to 100% confluent cells in two independent experiments. Automatic intensity measures were recorded from 4, 5 and 8 fields, for controls (C1 and C2), patients (P1, P2, LCA10_1 and LCA10_2) and MKS cells, respectively. C corresponds to C1 and C2 pooled values. ** P<0.01, ***P<0.001, ****P<0.0001; n.s., not significant; A.U., Arbitrary unit. Figure 7. View largeDownload slide Localization and abundance of CEP290 centriolar satellite partners. (A) Representative images of RAB8A (red) localization in the cilia from control (C1 and C2) and mutant (P1, P2, LCA10_1, LCA10_2 and MKS) fibroblasts induced to quiescence. ARL13B (green) labeling is used to mark the ciliary membrane. Image scale bar, 5 µm. Inset scale bar, 5 µm. (B) Quantification of RAB8A-positive cilia. Values are the mean±SEM (n>80; two independent experiments). C corresponds to C1 and C2 pooled values. ***P<0.001, ****P<0.0001; n.s., not significant; A.U., Arbitrary unit. Representative images of (C) CP110 or (E) PCM1 (green) centrosomal staining in quiescent control and mutant fibroblasts. Centrosomes are labeled by gamma-tubulin (γ-tub.; red). Image scale bar, 5 µm. Inset scale bar, 2 µm. Quantification of (D) CP110 or (F) PCM1 immunofluorescence intensity at centrosomes in quiescent fibroblasts. Values are the mean±SEM. Both immunolabelings were performed from 90 to 100% confluent cells in two independent experiments. Automatic intensity measures were recorded from 4, 5 and 8 fields, for controls (C1 and C2), patients (P1, P2, LCA10_1 and LCA10_2) and MKS cells, respectively. C corresponds to C1 and C2 pooled values. ** P<0.01, ***P<0.001, ****P<0.0001; n.s., not significant; A.U., Arbitrary unit. Figure 8. View largeDownload slide Size and abundance of cilia. (A) Representative images of cilia in the quiescent control (C1 and C2) and mutant (P1, P2, LCA10_1, LCA10_2 and MKS) fibroblasts. The cilium axoneme is stained with acetylated α-tubulin (Ac-tub; red) and the basal body is marked with pericentrin (PCN; green). Scale bar, 5 µm. (B, C) Ciliogenesis analysis. (B) Abundance of ciliated cells and (C) length of cilia axonemes in control and mutant fibroblasts. Values are the mean±SEM. A minimum of 120 ciliated cells from three biological replicates were considered for each cell lines. C corresponds to C1 and C2 pooled values. *P<0.05, **P<0.01, ****P<0.0001, n.s., not significant. Figure 8. View largeDownload slide Size and abundance of cilia. (A) Representative images of cilia in the quiescent control (C1 and C2) and mutant (P1, P2, LCA10_1, LCA10_2 and MKS) fibroblasts. The cilium axoneme is stained with acetylated α-tubulin (Ac-tub; red) and the basal body is marked with pericentrin (PCN; green). Scale bar, 5 µm. (B, C) Ciliogenesis analysis. (B) Abundance of ciliated cells and (C) length of cilia axonemes in control and mutant fibroblasts. Values are the mean±SEM. A minimum of 120 ciliated cells from three biological replicates were considered for each cell lines. C corresponds to C1 and C2 pooled values. *P<0.05, **P<0.01, ****P<0.0001, n.s., not significant. The trafficking of retinitis pigmentosa GTPase regulator, intraflagellar transport 25 and intraflagellar transport 88 is apparently normal in P1 and P2 fibroblasts To assess cilia trafficking in cells expressing CEP290 proteins lacking amino acid sequences encoded by Exons 18 and 8 and/or 32, we analyzed the subcellular localization of retinitis pigmentosa GTPase regulator (RPGR), intraflagellar transport (IFT) 25 and IFT88 by immunocytochemistry in quiescent mutant and control cells. Consistent with unaltered cilia trafficking, the subcellular localization of RPGR, IFT25 and IFT88 in P1 and P2 cell lines was comparable to that of LCA10_1, LCA10_2 and control individuals (Fig. 9). Figure 9. View largeDownload slide Intraflagellar trafficking. Representative images of (A) RPGR, (C) IFT25 or (E) IFT88 (green) localization in the cilium in quiescent control (C1 and C2) and mutant (P1, P2, LCA10_1, LCA10_2 and MKS) fibroblasts. The ciliary axoneme is marked by the glutamylated-tubulin (GT-335; red) or acetylated α-tubulin (Ac-tub.; red). Scale bar, 5 µm. Quantification of (B) RPGR-, (D) IFT25- or (F) IFT88-positive cilia. Values are the mean±SEM (n>80 for each condition; two independent experiments). C corresponds to C1 and C2 pooled values. ***P<0.001, ****P<0.0001, n.s., not significant. Figure 9. View largeDownload slide Intraflagellar trafficking. Representative images of (A) RPGR, (C) IFT25 or (E) IFT88 (green) localization in the cilium in quiescent control (C1 and C2) and mutant (P1, P2, LCA10_1, LCA10_2 and MKS) fibroblasts. The ciliary axoneme is marked by the glutamylated-tubulin (GT-335; red) or acetylated α-tubulin (Ac-tub.; red). Scale bar, 5 µm. Quantification of (B) RPGR-, (D) IFT25- or (F) IFT88-positive cilia. Values are the mean±SEM (n>80 for each condition; two independent experiments). C corresponds to C1 and C2 pooled values. ***P<0.001, ****P<0.0001, n.s., not significant. Discussion Here, we report on the identification of homozygosity and compound heterozygosity for CEP290 mutations predicted to truncate the protein in two unrelated adult individuals presenting with early-onset and severe retinal dystrophy but notable preservation of central vision over two to three decades. This presentation contrasts with the typical ophthalmologic phenotype described in individuals carrying biallelic CEP290 truncating mutations who present a congenital and dramatically severe retinal dystrophy with major central dysfunction, which is inconsistent with useful vision (3,7). Consistent with the predictive model of CEP290 disease pathogenesis, which suggests that the phenotype is correlated with the amount of full-length or minimally shortened functional protein that can be produced from mutant alleles (3), we observed that CEP290 proteins were produced in dermal cells from P1 and P2, despite biallelism for presumably truncating mutations. mRNA analysis suggests that these proteins arose from differential splicing of Exons 18, 8 and 32, producing minimally shortened mRNA isoforms with intact reading frames escaping NMD, contrary to full-length mutant mRNA. Previously, Drivas et al. reported that CEP290 Exons 6, 10, 41 and 46 undergo non-canonical BES in WT skin fibroblasts (3). Here, studying the same cell-type, we detected CEP290 isoforms lacking either Exon 18 or Exon 32 but not Exon 8. BES of Exon 18 is probably the primary mechanism that enables the production of a CEP290 protein in P1 cells by providing them with the ability to accumulate a minimally shortened isoform that has an intact reading frame. In addition to setting Exons 17 and 19 in the same frame, the c.1666del mutation may also contribute conferring a lower chance of skipping mutant Exon 17 than the WT. This could synergize with the skipping of the adjacent PTC-encoding Exon 18. Recently, pre-mRNA splicing of CEP290 in iPSC-derived optic cups has been shown to differ significantly from that observed in fibroblasts (13), questioning the relevance of spontaneous skipping of Exon 18 in dermal cells with respect to the retina. Interestingly, analyzing a WT human fetal retina, we detected CEP290 lacking Exon 18, the amount of which was significantly higher than its counterpart in fibroblasts, as was that of the full-length isoform. This PTC-containing isoform likely undergoes NMD as it does in dermal cells (Fig. 2A, D and G). Thus the level of Exon 18 BES in the target tissue cannot be known. Likewise, in the absence of P1 iPSC-derived optic cups, the amount of PTC-free CEP290 lacking Exon 18 cannot be determined. In contrast to the situation observed in P1 cells, BES could not account alone for the skipping of Exon 32 in fibroblasts from P2, which occurred at a far higher level than in controls. ESE sequences are usually purine- or A/C-rich (14,15) and reduced enhancer activity has been reported when a T is introduced into an artificial polypurine sequence, mimicking an ESE of the dystrophin gene, where the suppression of the enhancer is more pronounced when a T creates a nonsense codon compared with a missense codon (16). Interestingly, the c.4090G>T mutation in Exon 32 introduces a T in a purine-rich sequence, predicting an alteration of an ESE with increased chance of exon skipping, whereas the introduction of any other nucleotide at this position does not (Table 2). This suggests that the c.4090G>T variation participates in the significant elevation of the amount of CEP290 lacking Exon 32 in P2 cells compared with controls through a mechanism known as Class I NAS. This type of NAS, triggered by ESE disruption, contrasts with Class II NAS, which is elicited by the disruption of the reading-frame through a nuclear scanning mechanism that recognizes the reading frame of the pre-mRNA (17). Regarding Exon 8, the c.508A>T mutation, like the c.4090G>T variation in Exon 32, introduces a T in a purine-rich ESE sequence and increases the probability of exon skipping, whereas any other nucleotide change at this position does not (Table 2). Considering that we observed CEP290 lacking Exon 8 in P2 fibroblasts but not in controls and WT retina, it is likely that the skipping is owing to Class I NAS, contrasting with that of Exons 18 and 32 involving BES and BES with NAS, respectively. CEP290 is one among many proteins that localize at the centrosome, the main microtubule-organizing center in eukaryotes. While centrosomes participate in the cell cycle, upon the transition from proliferation to quiescence, they migrate toward the cell surface to initiate cilia formation (18). CEP290 is among the most important proteins in this process. It is a regulator of cilia formation through its interaction with PCM1, RAB8A and CP110, which are the major components of a functional network whose integrity is required for proper ciliation. CEP290 localizes to centriolar satellites and binds to PCM1, where it recruits RAB8A (11). CP110 binds to and antagonizes CEP290. Upon entry into quiescence, CP110 is released, allowing ciliation (10). CEP290 depletion has been reported to alter the centrosomal distribution of PCM1 (accumulation) and RAB8A (abrogation), leading to cilia formation defects. Depletion of CEP290 had no effect on CP110 localization and level, as determined by immunofluorescence and western blot analysis, respectively (10). In MKS cells where no CEP290 could be detected upon immunoblot analyzes, the PCM1 and RAB8A distribution was altered as expected and we observed a highly significant accumulation of CP110 at the centrosome as determined by quantitative immunofluorescence analysis (Fig. 7). In homozygous P1 cells, the abundance of the truncated CEP290 produced from the mRNA lacking Exon 18 was ∼10% that of the WT in controls (Fig. 4), whereas in compound heterozygous P2 cells, the amount of CEP290 isoforms produced from both skipped mRNA species detected by RT-PCR (i.e. lacking Exon 8 or 32) was significantly higher (30% of the WT in controls; P < 0.05; Fig. 4). The amount of a high molecular weight complex involving CEP290 was also likely reduced in P1 compared with P2 cells (Fig. 6). Both observations could be owing to differential expression of skipped mRNA in P1 and P2 cells, but this hypothesis cannot be verified since RT-qPCR does not allow comparison of different mRNA species in two or more samples. Alternatively, the CEP290 protein expressed in P1 cells lacking 38 residues of the central homo/heterodimerization domain encoded by Exon 18 could have reduced stability compared with the isoforms deleted of short regions of the NH2-terminal homo/heterodimerization and RAB8A interaction domains encoded by Exons 8 and 32, respectively (Fig. 3). In contrast, whether CEP290 in P1 cells has reduced interaction abilities is unlikely. If it does, this would probably alter the amount of CEP290-interacting proteins at centrosomes, as seen in P2 cells where moderate reduction in RAB8A staining is observed which could be reasonably attributed to the altered binding properties of the CEP290 isoform lacking residues encoded by Exon 32. Very interestingly, despite significantly reduced and variable levels of shortened CEP290 isoforms as determined by immunoblot analysis, the amount of CEP290 measured by immunofluorescence at the centrosomes was similar in P1 and P2 cells, reaching ∼50% of the WT and allowing normal ciliation. Together these observations support the view that (i) truncated CEP290 isoforms accumulate at the centrosome upon serum starvation, (ii) these isoforms conserve efficient interactions with key centrosomal proteins, including RAB8A whose minor depletion has no obvious effect on ciliation (Figs 5 and 7). These observations also suggest that the c.1666del, c.508A>T and c.4090G>T mutations could be regarded as hypomorphic variants. Interestingly, while to our knowledge homozygosity for the c.1666del has not been described previously, the change has been reported to cause Joubert syndrome (JS) in association with the c.3904C>T (p.Gln1302*), c.6031C>T (p.Arg2011*) or c.6012–12T>A mutations that affect Exons 31, 44 and intron 43, respectively (19–21). The c.6012–12T>A mutation abolishes the role of the natural acceptor splice site of intron 43 by activating a stronger neighboring cryptic splice site, the use of which introduces 56 nucleotides of the intronic sequence and a PTC in the mRNA. The c.3904C>T and c.6031C>T nonsense mutations confer to Exons 31 and 44 a higher chance of skipping compared with WT (Supplementary Material, Table S3). While the skipping of Exon 44 would disrupt the reading frame, that of Exon 31 would allow bypassing PTC. However, the encoded protein would lack a large stretch of 150 amino acids that are involved in RAB8A binding, presumably disrupting the RAB8A, PCM1 and CP110 functional complex involved in cilia formation (11). Hence, alleles found in combination with the c.1666del in JS are unlikely to encode functional CEP290, in accordance with the CEP290 predictive model of disease pathogenesis. Regarding Exon 32, unlike Exon 8, it has been involved in other individuals suffering from ciliopathies. The c.4090G>T change has been described in association with the c.3265C>T nonsense mutation (p.Gln1089*) in a LCA10 individual (22). This other nonsense mutation lies within Exon 28 that, if skipped, would produce another PTC, hence hampering the ability of cells in the individual to escape protein truncation. The c.4115–4116del introduces a PTC within Exon 32 (p.Ile1372Lysfs*5). Unlike the c.4090G>T mutation, it is predicted to reduce the chance of skipping Exon 32, thus producing no CEP290. The identification of this mutation in a LCA10 individual harboring the hypomorphic c.2991+1655A>G substitution and in an MKS fetus carrying the c.1219–1220del (p.Met407Glufs*14) in Exon 14, for which skipping would disrupt the reading frame, is consistent with this view. Intriguingly, while a low amount of minimally shortened CEP290 allowed apparently unaltered ciliation in the P1 and P2 cells, marked PCM1 redistribution and cilia defects were noted in LCA10_1 and LCA10_2, which expressed WT CEP290 (Figs 7E, F and 8). Of note cilia anomalies in LCA10_1 and LCA10_2 fibroblasts were less marked than the ones we reported earlier in the same cell lines [30% versus 50% of cells with absent cilium in (5)]. This variability might be essentially owing to the fact that we increased cell confluence in the present study [90–100% versus 80% in (5)]. Indeed, it has been reported that contact inhibition can induce quiescence and cilia formation even before serum-starvation (23). Extended serum-starvation in the present study [48 h versus 30 h in (5)] might also have influenced the kinetics of cilia formation. PCM1 redistribution and cilia formation alterations in LCA10_1 and LCA10_2 individuals raise the question of whether the c.2991+1655A>G allele produces a stable truncated protein that could interfere with the WT counterpart. The protein encoded by this allele would encompass the PCM1—but not the RAB8A—interacting domains. While CEP290 depletion has been reported to induce centrosomal PCM1 retention, the production of smaller size PCM1 complexes from the truncated protein could aggravate the phenomenon by altering the PCM1-dependent shuttling between the centrosome and the cytosol (11). Considering the ciliation anomalies, it is worth remembering that cellular expression of CEP290 fragments lacking the RAB8A domain of interaction have been shown to alter cilia formation (10). In summary, we report here naturally occurring exon skipping that produces CEP290 isoforms retaining functional interactions with centrosomal proteins important to cilia formation and intraflagellar trafficking, as the likely cause of unusually mild retinal disease in two unrelated individuals. While NAS has been previously suggested to account for atypical CEP290-related retinal diseases (8,9), our data suggest that BES can also contribute. Whatever the mechanism, the evidence of diminished severity in individuals producing low levels of minimally shortened protein supports the view that some CEP290 exons may be dispensable. This observation provides strong support of splice switching oligonucleotide-mediated therapies as a means to bypass protein truncation in patients with CEP290 mutations that introduce PTCs. Materials and Methods Patients Patient 1 (II: 2, Family 1), an 18-year-old woman, is the second of two children born to apparently unrelated parents of French origin. At the age of 18 months, she manifested uncomfortable mobility in dim light environments. Upon examination, she presented with hypermetropia (+4.5 and +5 diopters, right and left eyes, respectively), peripheral scotoma and extinct electroretinogram (ERG). Ophthalmoscopic examination was initially unremarkable, but retinal lesions were overt before teenage with multiple mid-peripheral white dots and cystoid macular edema at the fundus, cystic structures and severe thinning of the photoreceptor layer at optic coherence tomography and increased autofluorescence in the perimacular and inferior vascular arcade at autofluorescence imaging approximately 10, and peripheral osteoblast-like pigmentary deposits at 13 years of age, respectively. At the age of 18 years, the visual field was reduced to the central 10°, with some preserved peripheral islets and visual acuities of 20/40 and 20/30 (right and left eyes, respectively) (Table 1). The patient has received 200 mg acetazolamide daily since the age of 10 years old to treat her macular edema. Panel-based molecular diagnosis testing for inherited retinal diseases identified homozygosity for the CEP290 c.1666del (p.Ile556Phefs*17) mutation (Fig. 1A; Table 1). Patient 2 (II: 1, Family 2), a 33-year-old man, is the first of two children born to unrelated Portuguese parents. He presented at a genetic consultation with the Genetic Department of our hospital with a history of night blindness around the age of 2 years and a diagnosis of retinitis pigmentosa at the age of 7 years. Upon examination at the Ophthalmologic Department of our hospital at the age of 21 years, he presented with night blindness, a tubular visual field, fixation nystagmus, pure blue-yellow dyschromatopsia and visual acuities of 20/100 and 20/400 (right and left eyes, respectively). Fundoscopy showed absent macular reflex without rearrangement, dull retina without pigment migration at the fundus and thin retinal vessels. Examination at 31-year-old demonstrated no deterioration of the fundus aspect or of his visual acuities. The RD panel gene screening identified compound heterozygous CEP290 nonsense mutations c.508A>T (p.Lys170*) and c.4090G>T (p.Glu1364*) (Fig. 1B;Table 1). Familial analysis demonstrated biallelism and segregation of the mutations with disease in the two families (Fig. 1A and B). We also included in the study two individuals affected with LCA homozygously carrying the c.2991+1655A>G mutation (LCA10_1 and LCA10_2) and a fetus interrupted for MKS carrying the p.Arg205* change in homozygosity (MKS). These subjects were previously referred to as P1 and P2 (5), and Family 1_Subject 5 (24), respectively (Table 1). Written informed consent was obtained from all participating individuals or their legal representatives and the study was approved by the Comité de Protection des Personnes ‘Ile-De-France II’. Methods In silico analysis of mutations on splicing The effect on the splicing of the CEP290 mutations identified in the P1 and P2 patients was analyzed in silico using splice signal detection software {Human Splicing Finder, NNSPLICE from fruitfly.org, SpliceSiteFinder-like, MaxEntScan, GeneSplicer [Alamut Interpretation Software 2.0 (gateway for Human Splicing Finder, NNSPLICE, SpliceSiteFinder-like, MaxEntScan, GeneSplicer, ESEFinder, RESCUE-ESE and EX-SKIP), http://www.interactive-biosoftware.com; date last accessed May 22, 2018]}, ESE binding site detection software (ESEFinder, RESCUE-ESE), the EX-SKIP program, which compares the ESS/ESE profile of the WT, and a mutated allele to determine which exonic variant has the highest chance to skip this exon, and HOT-SKIP (HOT-SKIP program, http://hot-skip.img.cas.cz; date last accessed May 22, 2018), which systematically examines all possible substitutions in each exonic position that are most likely to skip the submitted exonic sequence. Primary cell culture Skin biopsies were obtained from the affected subjects: P1 (II: 2, Family 1), P2 (II: 1, Family 2), LCA10_1, LCA10_2, MKS and two control individuals (C1, C2) (Table 1). Primary fibroblasts were isolated by selective trypsinization and proliferated at 37°C with 5% CO2 in Opti-MEM Glutamax I medium (Life Technologies, Saint Aubin, France) supplemented with 10% fetal bovine serum (Life Technologies), 1% Ultroser-G serum substitute (Pall, Saint-Germain-en-Laye, France), 50 U/ml penicillin and 50 μg/ml streptomycin (Life Technologies). Cells were grown in 75 cm2 flasks (<12 passages) and cultured on a 0.1 mg/ml collagen support for fibroblasts from the MKS fetus. For the inhibition of nonsense-mediated mRNA decay, cells were seeded in a six-well plate before being incubated with 25 μg/ml of emetine dihydrochloride hydrate (Sigma-Aldrich, Saint-Quentin Fallavier, France) directly added to the fresh culture media for 15 h. RNA preparation and cDNA synthesis Total RNA from fibroblasts and WT human fetal (22 weeks) retina was extracted using the RNeasy Mini Kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s protocol. All samples were DNase-treated by the RNase-free DNase set (Qiagen). The concentration and purity of total RNA was determined using the Nanodrop-2000 spectrophotometer (ThermoScientific, Illkirch, France) before storage at −80°C. First-stranded cDNA synthesis was performed from 500 ng of total RNA extracted using the Verso cDNA kit (ThermoScientific) with random hexamer: anchored oligo(dT) primers at a 3:1 (vol:vol) ratio according to the manufacturer’s instructions. A non-reverse transcription reaction (without enzyme; RT-) for one sample was prepared to serve as a control for reverse transcription PCR (RT-PCR) and real-time quantitative PCR (RT-qPCR) experiments. RT-PCR CEP290 splicing isoforms were amplified from reverse transcribed mRNAs (2 µl) in 20 µl of 1× Phusion HF buffer containing 4 mM dNTPs (ThermoScientific), 0.4 units of Phusion High-Fidelity DNA polymerase (ThermoScientific) and 10 µM of specific primer pairs (Supplementary Material, Fig. S2 and Table S1). No template (NTC) reactions were used as negative controls. PCRs were carried out on a 2720 Thermal Cycler (Applied Biosystems, Courtaboeuf, France) under the following conditions: initial denaturation at 98°C for 5 min, followed by 32 cycles of 20 s denaturation at 98°C, 15 s annealing at 62°C and 30 s extension at 72°C. PCR products (10 µl) were separated by electrophoresis in a 3% low-melting agarose gel stained with ethidium bromide, visualized under UV lights and cut off. In-gel PCR products (2–5 μl; 65°C) were further sequenced using the Big Dye TerminatorCycle Sequencing Kit v3.1 (ABI Prism™, Applied Biosystems, Foster City, USA) on a 3500 automated sequencer (Applied Biosystems). RT-qPCR analysis The abundance of CEP290 mRNA isoforms was measured using primers specific to the unskipped (referred to as ‘full length’) or skipped CEP290 versions. Primer sequences are listed in Supplementary Material, Figure S2 and Table S2. GUSB (NM_000181.3), RPLP0 (NM_001002.3) mRNAs and the ALB (NM_000477) gene were used to normalize the data and control the non-contamination of cDNAs by genomic DNA, respectively (5). The cDNA (5 μl of a solution diluted at 1:25 in RNAse-free H2O) of each sample was subjected to PCR amplification in real-time in a buffer (20 μl) containing SYBR GREEN PCR Master Mix (Life Technologies) and 300 nM forward and reverse primers in the following conditions: activation of Taq polymerase and denaturation at 95°C for 10 min followed by 50 cycles of 15 s at 95°C, and 1 min at 60°C. The specificity of the amplified products was determined after the analysis of the melting curve carried out at the end of each amplification using one cycle at 95°C for 15 s, then a graded thermal increase of 60°C to 95°C for 20 min. The data analysis and methodology were performed as previously described (5). Western blot and blue native-polyacrylamide gel electrophoresis (BN-PAGE) analysis Cells were lysed on ice for 1 h by repeated homogenization in a low detergent lysis buffer containing phosphate buffered saline (PBS) 1×, 1% Triton, Halt™ Protease Inhibitor Cocktail 1X (ThermoScientific) and 25 U/ml Pierce Universal Nuclease (ThermoScientific). The lysates were centrifuged (20 000g at 4°C for 15 min), supernatants were collected and proteins quantified using the Bradford method. For western blot analysis, proteins (100 µg) were resolved by a 4–15% polyacrylamide gel (mini-PROTEAN TGX, Bio-Rad, Marnes-la-Coquette, France) according to the supplier’s recommendations. All lysates were heated at 95°C for 10 min prior to loading. Proteins were transferred to a PVDF 0.2 µM membrane (Bio-Rad) using a Trans-Blot Turbo Transfer System (Bio-Rad), and then processed for immunoblotting. Membranes were probed with polyclonal rabbit anti-C-terminal-CEP290 (1:1500, Novus Biologicals, USA) and monoclonal mouse anti-β-actin (1:2000, Abcam, Paris, France) primary antibodies, and then incubated with donkey anti-rabbit IgG-HRP (1:2000, ThermoScientific) and donkey anti-mouse IgG-HRP secondary antibodies (1:4000, ThermoScientific), respectively. Blots were developed with the use of the Clarity Western ECL Substrate (Bio-Rad) and ChemiDoc XRS+ Imaging System (Bio-Rad). Western blot images were acquired and analyzed with Image Lab software 3.0.1 build 18 (Bio-Rad). The abundance of CEP290 relative to β-actin was estimated in each cell line by densitometry using Image Lab software (Bio-Rad). CEP290 abundance levels are the means of five independent protein extracts. For BN-PAGE, the proteins (100 µg) were solubilized by 250 U/µl Pierce Universal Nuclease (ThermoScientific) and loaded on a native 4–16% Bis-Tris gel (Life Technologies). Native proteins were transferred to a PVDF membrane (Merck-Millipore, Fontenay sous Bois, France) overnight at 25 V and 4°C in transfer buffer containing 0.02% SDS, and processed for immunoblotting as described above. Protein complexes containing Vinculin were immunolabeled by rabbit anti-Vinculin antibody (1:10 000; Abcam) to serve as the loading control. OXPHOS proteins from isolated mitochondrial membranes (20 µg) were solubilized by 2% lauryl maltoside (25), were loaded and complex 1 of the respiratory chain was immunolabeled using an anti-GRIM19 antibody (1:10 000; Abcam) to serve as the high molecular weight (1 MDa) marker. Immunocytochemistry analysis Cells were grown on glass coverslips in a 12-well plate before being incubated for 48 h in serum-free medium. After serum-starvation, the cells were fixed with cold methanol (7 min at –20°C) and washed two times with PBS. Cells were permeabilized and nonspecific sites were saturated in a PBS solution containing 5% normal goat serum, 3% bovine serum albumin and 0.5% Triton X-100 for 1 h. Permeabilized cells were incubated overnight at 4°C with primary antibodies in PBS containing 3% bovine serum albumin and 0.1% Triton X-100:anti-Pericentrin rabbit antibody (1:1000, Abcam), anti-γ-tubulin mouse antibody (1:500, Sigma-Aldrich), anti-CEP290 rabbit antibody (1:100, Novus Biologicals), anti-CP110 rabbit antibody (1:100, ProteinTech), anti-PCM1 rabbit antibody (1:100, Cell Signaling Technology), anti-acetylated α-Tubulin mouse antibody (1:1000, Sigma-Aldrich), anti-GT335 mouse antibody (1:100, Adipogen), anti-ARL13B rabbit antibody (1:100, ProteinTech), anti-IFT25 rabbit antibody (1:100, ThermoScientific) anti-IFT88 rabbit antibody (1:100, ProteinTech), anti-RPGR rabbit antibody (1:100, Sigma-Aldrich) and anti-RAB8A mouse antibody (1:50, Abnova). After three washes in PBS, the cells were incubated for 1 h at room temperature with secondary antibodies in PBS solution containing 3% bovine serum albumin and 0.1% Triton X-100:anti-rabbit IgG goat antibody coupled to Alexa-Fluor 488 and anti-mouse IgG goat antibody coupled to Alexa-Fluor 568 (1:1000, Life Technologies). After three additional washes with PBS, the coverslips were mounted on slides using a mounting medium containing DAPI (ProLong Gold antifade reagent with DAPI, Invitrogen) to stain the cell nuclei. Immunofluorescence images were obtained using a Zeiss LSM700 confocal microscope. Exposure times and settings for image processing were constant for all samples to allow sample comparison. The number of Z-stacks collected was variable between the samples but optimized for capturing maximum fluorescent signals. Deconvoluted images were projected into one picture using ImageJ software (https://imagej.nih.gov/ij; date last accessed May 22, 2018) Z-project tool with the maximum intensity setting. The total fluorescence contained in 7 and 16 µm2 squares centered on the centrosome as determined by γ-tubulin staining was recorded to measure the intensity of CEP290 and CP110 and that of the PCM1 labelling in the peri-centrosomal region, respectively. Integrated pixel densities were quantified in each square using ImageJ software and a default threshold for background subtraction of 30. The final images were generated using ImageJ software. Statistics The data obtained from C1 and C2 were systematically pooled. Prism6 software was used for statistical analyses. The significance of variation among samples was determined using one-way ANOVA with a post hoc Dunnett’s test in all experiments but RT-qPCR for which we used a post hoc Tukey’s test. Error bars reflect the standard error of the mean (SEM). Supplementary Material Supplementary Material is available at HMG online. Acknowledgements We gratefully acknowledge the patients that donated skin biopsies for this study. Conflict of Interest statement. None declared. Funding This work was supported by Retina France, by grants from the Fondation JED-Belgique to X.G., UNADEV-AVIESAN/ITMO NNP (R16074KS) to I.P. and UNADEV-AVIESAN/ITMO NNP (R16073KS) to J.-M.R. References 1 Ware S.M. , Aygun M.G. , Hildebrandt F. ( 2011 ) Spectrum of clinical diseases caused by disorders of primary cilia . Proc. Am. Thorac. Soc ., 8 , 444 – 450 . Google Scholar CrossRef Search ADS PubMed 2 Papon J.F. , Perrault I. , Coste A. , Louis B. , Gérard X. , Hanein S. , Fares-Taie L. , Gerber S. , Defoort-Dhellemmes S. , Vojtek A.M. et al. ( 2010 ) Abnormal respiratory cilia in non-syndromic Leber congenital amaurosis with CEP290 mutations . J. Med. Genet ., 47 , 829 – 834 . 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For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Human Molecular Genetics Oxford University Press

Basal exon skipping and nonsense-associated altered splicing allows bypassing complete CEP290 loss-of-function in individuals with unusually mild retinal disease

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Abstract

Abstract CEP290 mutations cause a spectrum of ciliopathies from Leber congenital amaurosis type 10 (LCA10) to embryo-lethal Meckel syndrome (MKS). Using panel-based molecular diagnosis testing for inherited retinal diseases, we identified two individuals with some preserved vision despite biallelism for presumably truncating CEP290 mutations. The first one carried a homozygous 1 base pair deletion in Exon 17, introducing a premature termination codon (PTC) in Exon 18 (c.1666del; p.Ile556Phefs*17). mRNA analysis revealed a basal exon skipping (BES) of Exon 18, providing mutant cells with the ability to escape protein truncation, while disrupting the reading frame in controls. The second individual harbored compound heterozygous nonsense mutations in Exon 8 (c.508A>T, p.Lys170*) and Exon 32 (c.4090G>T, p.Glu1364*), respectively. Some CEP290 lacking Exon 8 were detected in mutant fibroblasts but not in controls whereas some skipping of Exon 32 occurred in both lines, but with higher amplitude in the mutant. Considering that the deletion of either exon maintains the reading frame in either line, skipping in mutant cells likely involves nonsense-associated altered splicing alone (Exon 8), or with BES (Exon 32). Skipping of PTC-containing exons in mutant cells allowed production of CEP290 isoforms with preserved ability to assemble into a high molecular weight complex and to interact efficiently with proteins important for cilia formation and intraflagellar trafficking. In contrast, studying LCA10 and MKS fibroblasts we show moderate to severe cilia alterations, providing support for a correlation between disease severity and the ability of cells to express shortened, yet functional, CEP290 isoforms. Introduction Ciliopathies are a large group of Mendelian disorders caused by direct or indirect dysfunction of primary or motile cilia found on most vertebrate cells (1). In humans, ciliopathies are characterized by prominent genetic heterogeneity and pleiotropy. Mutations in CEP290 (MIM610142, https://www.omim.org; date last accessed May 22, 2018), encoding the 290-kDa centrosomal protein, have been involved in a large spectrum of ciliopathies, ranging from isolated retinal dystrophy to multi-visceral and sometimes embryo-lethal Meckel syndrome type 4 (MKS; MIM611134) (2). It has been proposed that the origin of CEP290 pleiotropy lies not in the effects of mutations on protein function, but rather in the effects of mutations on the amount of protein retaining all or some of the full-length CEP290 functionality, which can be supplied to the cell (3). Consistent with this view, isolated retinal disease has been ascribed to hypomorphic mutations, the most frequent of which is a deep intronic change c.2991+1655A>G, which introduces a cryptic exon and a premature termination codon (PTC) in 50–75% of the CEP290 mRNA products (4,5). Other hypomorphic variants have been reported to preferentially cluster in exons, which experience spontaneous non-canonical skipping through a mechanism referred to as CEP290 basal exon skipping (BES), which likely enables the production of low levels of near-full length functional protein from mutation-free mRNA with intact reading frames (3). The CEP290-retinal disease typically presents as a neonatal-onset and dramatically severe cone-rod dystrophy, known as Leber congenital amaurosis (LCA) type I (6,7) (MIM24000). The report of selective exclusion of a CEP290 exon with a stop codon in individuals with unexpectedly mild retinal disease (8,9) has suggested that nonsense-associated altered splicing (NAS) may be another mechanism and, in the light of the attenuated phenotype, likely a more efficient one, that provides cells the potential to compensate for deleterious mutations. However, no analysis supported this hypothesis. Using gene panel-based sequencing in a series of individuals with retinal dystrophies (RD) of variable presentation, we found homozygosity for a one base pair (bp) deletion and compound heterozygosity for nonsense mutations in two unrelated adult individuals with unusually preserved central vision. Here, we report that, despite biallelism for apparent truncating mutations, fibroblasts from the patients produced minimally shortened CEP290 proteins, which retained functional interactions important to cilia formation and intraflagellar trafficking. We show that BES and/or NAS of exons allowed bypassing PTC in the cells from the two individuals. Results Panel-based molecular diagnosis testing identifies biallelism for presumably truncating CEP290 mutations in two individuals (P1 and P2) affected with an unusually moderate retinal disease Analyzing a molecular diagnosis panel of 190 genes for inherited retinal diseases in a large series individuals affected with RD of variable age, mode of onset and clinical presentation (article in preparation), we identified homozygosity and compound heterozygosity for presumably truncating CEP290 mutations in two unrelated individuals (P1 and P2): a 1 bp deletion in Exon 17 predicted to introduce a PTC in Exon 18 and nonsense mutations in Exon 8 (c.508A>T, p.Lys170*) and Exon 32 (c.4090G>T, p.Glu1364*), respectively (Fig. 1). In contrast with CEP290 genotype–phenotype correlations (4,7), the two individuals had notable preservation of central vision over two to three decades despite early-onset and severe retinal dystrophy (Table 1). Table 1. Mutations and clinical features of individuals included in the study Name Agea (year) CEP290 mutations Phenotype References Control-1; C1 8 None No overt pathology – Control-2; C2 10 None No overt pathology – Patient II: 2 (family 1) P1 18 c.1666del (p.Ile556Phefs*17) c.1666del (p.Ile556Phefs*17) EOSRD No nystagmus, no photophobia Photo-attraction, night discomfort Scotopic and photopic ERG non-recordable Visual acuity: RE=20/40, LE=20/30 – Patient II: 1 (family 2) P2 33 c.508A>T (p.Lys170*) c.4090G>T (p.Glu1364*) EOSRD No nystagmus, no photophobia Photo-attraction, night discomfort Scotopic and photopic ERG non-recordable Visual acuity: RE=20/100, LE=20/400 – LCA10_1 3 c.2991 + 1655A>G (p.Cys998*) c.2991 + 1655A>G (p.Cys998*) LCA Nystagmus, photophobia Scotopic and photopic ERG non-recordable No visual acuity (5) LCA10_2 33 c.2991 + 1655A>G (p.Cys998*) c.2991 + 1655A>G (p.Cys998*) LCA Nystagmus, photophobia Scotopic and photopic ERG non-recordable No visual acuity (5) MKS Embryo c.613C>T (p.Arg205*) c.613C>T (p.Arg205*) MKS (24) Name Agea (year) CEP290 mutations Phenotype References Control-1; C1 8 None No overt pathology – Control-2; C2 10 None No overt pathology – Patient II: 2 (family 1) P1 18 c.1666del (p.Ile556Phefs*17) c.1666del (p.Ile556Phefs*17) EOSRD No nystagmus, no photophobia Photo-attraction, night discomfort Scotopic and photopic ERG non-recordable Visual acuity: RE=20/40, LE=20/30 – Patient II: 1 (family 2) P2 33 c.508A>T (p.Lys170*) c.4090G>T (p.Glu1364*) EOSRD No nystagmus, no photophobia Photo-attraction, night discomfort Scotopic and photopic ERG non-recordable Visual acuity: RE=20/100, LE=20/400 – LCA10_1 3 c.2991 + 1655A>G (p.Cys998*) c.2991 + 1655A>G (p.Cys998*) LCA Nystagmus, photophobia Scotopic and photopic ERG non-recordable No visual acuity (5) LCA10_2 33 c.2991 + 1655A>G (p.Cys998*) c.2991 + 1655A>G (p.Cys998*) LCA Nystagmus, photophobia Scotopic and photopic ERG non-recordable No visual acuity (5) MKS Embryo c.613C>T (p.Arg205*) c.613C>T (p.Arg205*) MKS (24) a Age at which the dermal biopsy and the last clinical examination were performed. EOSRD, early onset and severe retinal dystrophy; LCA, Leber congenital amaurosis; MKS, Meckel syndrome; ERG, electroretinogram. Table 1. Mutations and clinical features of individuals included in the study Name Agea (year) CEP290 mutations Phenotype References Control-1; C1 8 None No overt pathology – Control-2; C2 10 None No overt pathology – Patient II: 2 (family 1) P1 18 c.1666del (p.Ile556Phefs*17) c.1666del (p.Ile556Phefs*17) EOSRD No nystagmus, no photophobia Photo-attraction, night discomfort Scotopic and photopic ERG non-recordable Visual acuity: RE=20/40, LE=20/30 – Patient II: 1 (family 2) P2 33 c.508A>T (p.Lys170*) c.4090G>T (p.Glu1364*) EOSRD No nystagmus, no photophobia Photo-attraction, night discomfort Scotopic and photopic ERG non-recordable Visual acuity: RE=20/100, LE=20/400 – LCA10_1 3 c.2991 + 1655A>G (p.Cys998*) c.2991 + 1655A>G (p.Cys998*) LCA Nystagmus, photophobia Scotopic and photopic ERG non-recordable No visual acuity (5) LCA10_2 33 c.2991 + 1655A>G (p.Cys998*) c.2991 + 1655A>G (p.Cys998*) LCA Nystagmus, photophobia Scotopic and photopic ERG non-recordable No visual acuity (5) MKS Embryo c.613C>T (p.Arg205*) c.613C>T (p.Arg205*) MKS (24) Name Agea (year) CEP290 mutations Phenotype References Control-1; C1 8 None No overt pathology – Control-2; C2 10 None No overt pathology – Patient II: 2 (family 1) P1 18 c.1666del (p.Ile556Phefs*17) c.1666del (p.Ile556Phefs*17) EOSRD No nystagmus, no photophobia Photo-attraction, night discomfort Scotopic and photopic ERG non-recordable Visual acuity: RE=20/40, LE=20/30 – Patient II: 1 (family 2) P2 33 c.508A>T (p.Lys170*) c.4090G>T (p.Glu1364*) EOSRD No nystagmus, no photophobia Photo-attraction, night discomfort Scotopic and photopic ERG non-recordable Visual acuity: RE=20/100, LE=20/400 – LCA10_1 3 c.2991 + 1655A>G (p.Cys998*) c.2991 + 1655A>G (p.Cys998*) LCA Nystagmus, photophobia Scotopic and photopic ERG non-recordable No visual acuity (5) LCA10_2 33 c.2991 + 1655A>G (p.Cys998*) c.2991 + 1655A>G (p.Cys998*) LCA Nystagmus, photophobia Scotopic and photopic ERG non-recordable No visual acuity (5) MKS Embryo c.613C>T (p.Arg205*) c.613C>T (p.Arg205*) MKS (24) a Age at which the dermal biopsy and the last clinical examination were performed. EOSRD, early onset and severe retinal dystrophy; LCA, Leber congenital amaurosis; MKS, Meckel syndrome; ERG, electroretinogram. Figure 1. View largeDownload slide Identification of CEP290 truncating mutations in two affected individuals with unusually preserved central vision. (A, B) Pedigrees, segregation analysis and Sanger sequencing chromatograms of DNA showing the CEP290 mutations in Families 1 and 2. Filled and unfilled symbols represent affected and unaffected members, respectively. The affected individual P1 (II: 2, Family 1) is homozygous for the c.1666del (p.Ile556Phefs*17) mutation, whereas the affected individual P2 (II: 1, Family 2) is compound heterozygous for the c.508A>T (p.Lys170*) and c.4090G>T (p.Glu1364*) mutations. The biparental transmission is confirmed by the detection of mutations in single heterozygosity in parental DNAs. Red arrows indicate mutant nucleotide positions. Symbol+represents the WT allele. Figure 1. View largeDownload slide Identification of CEP290 truncating mutations in two affected individuals with unusually preserved central vision. (A, B) Pedigrees, segregation analysis and Sanger sequencing chromatograms of DNA showing the CEP290 mutations in Families 1 and 2. Filled and unfilled symbols represent affected and unaffected members, respectively. The affected individual P1 (II: 2, Family 1) is homozygous for the c.1666del (p.Ile556Phefs*17) mutation, whereas the affected individual P2 (II: 1, Family 2) is compound heterozygous for the c.508A>T (p.Lys170*) and c.4090G>T (p.Glu1364*) mutations. The biparental transmission is confirmed by the detection of mutations in single heterozygosity in parental DNAs. Red arrows indicate mutant nucleotide positions. Symbol+represents the WT allele. In silico analysis suggests that CEP290 mutations carried by P1 and P2 affect splicing The apparent inconsistency between phenotypes and genotypes in P1 and P2 individuals led us to analyze the effect of their mutations on splicing. The use of prediction software solutions analyzing splice signals and exonic splicing silencer (ESS)/exonic splicing enhancer (ESE) binding sites, suggests that the c.1666del identified in P1 in homozygosity does not modify significant consensus splice site scores. However, it decreases Exon 17 ESS/ESE ratio, thus reducing the susceptibility of skipping compared with the wild-type (WT) sequence (Table 2). The c.508A>T and c.4090G>T changes identified in compound heterozygosity in P2 do not modify the consensus splice site strength either. However, in contrast to the c.1666del, they increased the ESS/ESE ratios, conferring mutant Exons 8 and 32 with a higher chance of skipping than WT counterparts (Table 2). Additionally, the c.4090G>T allele is expected to create a strong donor splice-site 60 bp downstream of the start of Exon 32 (Supplementary Material, Fig. S1). Table 2. Impact of mutations on ESS and ESE motifs EX-SKIP predictions HOT-SKIP predictions Nucleotide ESS ESE ESS/ESE ESS ESE ESS/ESE Skipping predictions of mutant allele compared with WT allele c.508 Exon 8 A 0 21 0 0 10 0 – T 1 13 0.08 1 2 0.5 Higher chance G 0 20 0 0 9 0 Comparable chance C 0 24 0 0 13 0 Comparable chance c.1666 exon17 A 16 97 0.16 N.A. – delA 14 99 0.14 Lower chance c.4090 Exon 32 A 9 66 0.14 1 7 0.14 Lower chance T 12 60 0.20 4 1 4 Higher chance G 10 64 0.16 2 5 0.4 – C 8 59 0.14 0 0 0 Lower chance EX-SKIP predictions HOT-SKIP predictions Nucleotide ESS ESE ESS/ESE ESS ESE ESS/ESE Skipping predictions of mutant allele compared with WT allele c.508 Exon 8 A 0 21 0 0 10 0 – T 1 13 0.08 1 2 0.5 Higher chance G 0 20 0 0 9 0 Comparable chance C 0 24 0 0 13 0 Comparable chance c.1666 exon17 A 16 97 0.16 N.A. – delA 14 99 0.14 Lower chance c.4090 Exon 32 A 9 66 0.14 1 7 0.14 Lower chance T 12 60 0.20 4 1 4 Higher chance G 10 64 0.16 2 5 0.4 – C 8 59 0.14 0 0 0 Lower chance Effect of nucleotide changes at positions c.508, c.1666 and c.4090 on ESS and ESE motifs according to EX-SKIP and HOT-SKIP prediction programs. The WT and mutant alleles identified in this study are labeled in blue and red, respectively. N.A., not applicable; ESS, exonic splicing silencer; ESE, exonic splicing enhancer. Table 2. Impact of mutations on ESS and ESE motifs EX-SKIP predictions HOT-SKIP predictions Nucleotide ESS ESE ESS/ESE ESS ESE ESS/ESE Skipping predictions of mutant allele compared with WT allele c.508 Exon 8 A 0 21 0 0 10 0 – T 1 13 0.08 1 2 0.5 Higher chance G 0 20 0 0 9 0 Comparable chance C 0 24 0 0 13 0 Comparable chance c.1666 exon17 A 16 97 0.16 N.A. – delA 14 99 0.14 Lower chance c.4090 Exon 32 A 9 66 0.14 1 7 0.14 Lower chance T 12 60 0.20 4 1 4 Higher chance G 10 64 0.16 2 5 0.4 – C 8 59 0.14 0 0 0 Lower chance EX-SKIP predictions HOT-SKIP predictions Nucleotide ESS ESE ESS/ESE ESS ESE ESS/ESE Skipping predictions of mutant allele compared with WT allele c.508 Exon 8 A 0 21 0 0 10 0 – T 1 13 0.08 1 2 0.5 Higher chance G 0 20 0 0 9 0 Comparable chance C 0 24 0 0 13 0 Comparable chance c.1666 exon17 A 16 97 0.16 N.A. – delA 14 99 0.14 Lower chance c.4090 Exon 32 A 9 66 0.14 1 7 0.14 Lower chance T 12 60 0.20 4 1 4 Higher chance G 10 64 0.16 2 5 0.4 – C 8 59 0.14 0 0 0 Lower chance Effect of nucleotide changes at positions c.508, c.1666 and c.4090 on ESS and ESE motifs according to EX-SKIP and HOT-SKIP prediction programs. The WT and mutant alleles identified in this study are labeled in blue and red, respectively. N.A., not applicable; ESS, exonic splicing silencer; ESE, exonic splicing enhancer. RT-PCR analysis shows selective exclusion of PTC-containing exons in fibroblasts from P1 and P2 individuals Analysis of CEP290 mRNA from the patient and control skin-fibroblasts were consistent with the in silico predictions. Agarose gel analysis and Sanger sequencing of RT-PCR products generated from P1 mRNA using primers specific to Exons 15 and 19 (Supplementary Material, Fig. S2 and Table S1) detected a full-length isoform, which carried the c.1666del in Exon 17, and a shorter mRNA lacking Exon 18 (skipped isoform) (Fig. 2A andSupplementary Material, Fig. S3A). The c.1666del in the full-length mutant mRNA is predicted to disrupt the reading frame and introduce a PTC in Exon 18 (p.Ile556Phefs*17). Interestingly, in the skipped mRNA, the deletion of Exon 18 in combination with the 1 bp deletion in Exon 17 allows for maintaining an intact reading frame (Fig. 2A). In control fibroblasts, very low levels of CEP290 cDNA lacking Exon 18 could be detected, suggesting that this exon undergoes BES (Fig. 2A). Unlike the mutant, WT Exon 17 is not in frame with Exon 19 (Fig. 3). Thus, the skipping of Exon 18 introduces a PTC. Figure 2. View largeDownload slide Naturally occurring exclusion of CEP290 exons encompassing premature stop codon. (A–C) Analysis of reverse transcribed CEP290 mRNA extracted from human fetal retina (Retina), patient (P1 and P2) and control (C1) fibroblasts. Images of agarose gel showing RT-PCR fragments produced using primer pairs surrounding mutant Exons 17, 8 and 32, respectively. White asterisks point to heteroduplex products. The boxes summarize the exonic organization and phasing of each RT-PCR product. Red arrows show the position of the PTC within CEP290 isoforms. Numbers next to boxes refer to corresponding Sanger sequencing chromatograms represented in Supplementary Material, Figure S3. (D–F) Relative expression of WT (gray bars) and mutant (black bars) full-length isoforms, and (G–I) skipped (CEP290Δ18, CEP290Δ8 and CEP290Δ32; hatched bars) CEP290 mRNAs in human fetal retina (Retina), control (C1 and C2) and patient (P1 and P2) fibroblasts as determined by RT-qPCR using GUSB and RPLP0 genes as reference. C corresponds to C1 and C2 pooled values. Values are the mean±SEM derived from three independent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n.s., not significant. Figure 2. View largeDownload slide Naturally occurring exclusion of CEP290 exons encompassing premature stop codon. (A–C) Analysis of reverse transcribed CEP290 mRNA extracted from human fetal retina (Retina), patient (P1 and P2) and control (C1) fibroblasts. Images of agarose gel showing RT-PCR fragments produced using primer pairs surrounding mutant Exons 17, 8 and 32, respectively. White asterisks point to heteroduplex products. The boxes summarize the exonic organization and phasing of each RT-PCR product. Red arrows show the position of the PTC within CEP290 isoforms. Numbers next to boxes refer to corresponding Sanger sequencing chromatograms represented in Supplementary Material, Figure S3. (D–F) Relative expression of WT (gray bars) and mutant (black bars) full-length isoforms, and (G–I) skipped (CEP290Δ18, CEP290Δ8 and CEP290Δ32; hatched bars) CEP290 mRNAs in human fetal retina (Retina), control (C1 and C2) and patient (P1 and P2) fibroblasts as determined by RT-qPCR using GUSB and RPLP0 genes as reference. C corresponds to C1 and C2 pooled values. Values are the mean±SEM derived from three independent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n.s., not significant. Figure 3. View largeDownload slide Human CEP290 exon map and phasing. Schematic representation of the 54 exons of the human CEP290 gene. The black box corresponds to non-coding Exon 1. The start codon (ATG) in Exon 2 is represented by an arrow and the stop codon (TAA) is indicated at the end of Exon 54. Light gray boxes represent exons for which skipping would not disrupt the reading frame. Dark gray boxes indicate exons for which skipping would disrupt the reading frame. Protein interaction domains are represented above and/or below the sequence [based on references (26,27)]. Figure 3. View largeDownload slide Human CEP290 exon map and phasing. Schematic representation of the 54 exons of the human CEP290 gene. The black box corresponds to non-coding Exon 1. The start codon (ATG) in Exon 2 is represented by an arrow and the stop codon (TAA) is indicated at the end of Exon 54. Light gray boxes represent exons for which skipping would not disrupt the reading frame. Dark gray boxes indicate exons for which skipping would disrupt the reading frame. Protein interaction domains are represented above and/or below the sequence [based on references (26,27)]. Considering P2 fibroblasts, RT-PCR using primers specific to Exons 6 and 10, and 31 and 34 (Supplementary Material, Fig. S2 and Table S1), generated three and four PCR products, the sequencing of which identified (Supplementary Material, Fig. S3B and C): mutant (c.508A) and WT (c.508T) sequences encompassing Exons 6–10, an isoform lacking Exon 8, mutant (c.4090T) and WT (c.4090G) sequences encompassing Exons 31–34, an isoform lacking Exon 32 and another lacking the last 106 nucleotides of Exon 32, respectively (Fig. 2B and C). The complete deletion of Exon 8 or 32 removes the PTC, while maintaining the reading frame (Fig. 3). In contrast, the partial deletion of Exon 32 removes the c.4090G>T nonsense mutation yet disrupts the reading frame and introducing another PTC (p.Glu1364Phefs*20; Fig. 2C;Supplementary Material, Fig. S3C). In the controls, we detected the full-length CEP290 mRNA, but not the isoform deleted of Exon 8 (Fig. 2B). Considering that the skipping of WT Exon 8 would produce an in-frame NMD-resistant isoform, this observation suggests that this exon does not undergo BES in fibroblasts and that CEP290 lacking Exon 8 in P2 cells arose exclusively from the mutant allele. In contrast, we observed some Exon 32 skipping in the controls, yet with a significantly lower amplitude than in P2 cells. Because the deletion of this exon does not modify the reading frame in either cell lines, this observation suggests that both BES and NAS occurred in P2 cells (Fig. 2B and C). Analysis of mRNA from human retina supports BES of Exon 18 and 32 as in fibroblasts Consistent with what we observed in fibroblasts, agarose gel analysis and Sanger sequencing of RT-PCR products generated from human retina mRNA using primers flanking Exons 8, 18 and 32 (Supplementary Material, Fig. S2; Table S1) detected full-length CEP290 and mRNA isoforms lacking either Exon 18 or 32 but not Exon 8 (Fig. 2A–C;Supplementary Material, Fig. S3). RT-qPCR analysis supports NMD of PTC-encoding isoforms We determined the abundance of CEP290 in patient and control fibroblasts and in the retina by RT-qPCR using primers specific to full-length mRNAs and all skipped isoforms, except for the one partially deleted of Exon 32 (Supplementary Material, Fig. S2 and Table S2). This analysis revealed moderate amounts of CEP290 isoforms lacking either Exon 8, 18 or 32 in fibroblasts from P1 and P2 individuals (Fig. 2G–I). Since these isoforms have intact open reading frames free of PTC, this observation suggests that only reduced numbers of pre-mRNA copies underwent skipping in the fibroblasts from patients. In addition, we observed an under-representation of CEP290 isoforms encoding PTCs (full-length mutant mRNAs in P1 and P2 fibroblasts and mRNAs lacking Exon 18 in controls), compared with their counterparts, which have intact reading frames (full-length mRNAs in controls, skipped mRNAs in P1) (Fig. 2D–G). Consistent with the NMD of PTC-containing isoforms, their abundance significantly increased when fibroblasts were treated using an NMD inhibitor (emetine: 25 μg/ml, 15 h) (Supplementary Material, Fig. S4). In the retina, the full-length CEP290 mRNA was significantly more abundant than in control fibroblasts, supporting a higher CEP290 expression in the retina (4- to 5-fold) (Fig. 2D and F). Consistently, both the PTC-containing and PTC-free isoforms lacking Exons 18 and 32 were more abundant in the retina than in control fibroblasts (Fig. 2G and I). P1 and P2 fibroblasts express a CEP290 protein that localizes at the centrosome Protein lysates from 90 to 100% confluent mutant and control fibroblasts were analyzed by western blot using antibodies that recognize the carboxy-terminus extension of CEP290 and β-actin, respectively. We observed a band ∼290 kDa in P1 and P2 lysates as in controls, LCA10_1 and LCA10_2, and the abundance relative to β-actin of which varied strikingly (Fig. 4). In contrast, CEP290 was undetectable in MKS cells (Fig. 4A). Immunocytochemistry in 90–100% confluent serum-starved cells revealed CEP290 staining at the centrosome in P1 and P2 cells (Fig. 5A and B) with mean immunofluorescence intensities ∼30% of that of the controls (Fig. 5C). A comparable staining was observed in cells from LCA10_1 and LCA10_2 individuals (Fig. 5). These results are consistent with the view that, like WT CEP290, mRNAs deleted from Exons 18 and 8 and/or 32 produce minimally shortened, yet stable, CEP290 proteins, which have the ability to localize at the centrosome. Interestingly, although western blot analysis did not detect CEP290, immunocytochemistry disclosed some CEP290 positive MKS cells (Fig. 5). Figure 4. View largeDownload slide Effect of the selective exclusion of PTC-encoding exons on CEP290 protein production. (A) Immunodetection of the CEP290 protein in mutant fibroblasts (P1, P2, LCA10_1, LCA10_2 and MKS) and control cell lines (C1 and C2). β-Actin was used for normalization. (B) Quantification of CEP290 protein abundance. C corresponds to C1 and C2 pooled values. Values were determined by computed-densitometry analysis of CEP290 and β-actin expression in each sample and are the mean±SEM derived from five independent experiments. Whereas we measured a significant difference in CEP290 abundance between P1 and P2, there is none for LCA10_1 and LCA10_2 samples. *P<0.05, ****P<0.0001; n.s., not significant. Figure 4. View largeDownload slide Effect of the selective exclusion of PTC-encoding exons on CEP290 protein production. (A) Immunodetection of the CEP290 protein in mutant fibroblasts (P1, P2, LCA10_1, LCA10_2 and MKS) and control cell lines (C1 and C2). β-Actin was used for normalization. (B) Quantification of CEP290 protein abundance. C corresponds to C1 and C2 pooled values. Values were determined by computed-densitometry analysis of CEP290 and β-actin expression in each sample and are the mean±SEM derived from five independent experiments. Whereas we measured a significant difference in CEP290 abundance between P1 and P2, there is none for LCA10_1 and LCA10_2 samples. *P<0.05, ****P<0.0001; n.s., not significant. Figure 5. View largeDownload slide CEP290 expression in quiescent cells. (A) Representative images of CEP290 (green) localization in control and mutant fibroblasts induced to quiescence. Acetylated α-tubulin (Ac-tub; red) is used to mark the ciliary axoneme. As in control cell lines (C1 and C2), CEP290 is correctly localized at the base of the cilia in patient (P1 and P2) and LCA10 (LCA10_1 and LCA10_2) fibroblasts. CEP290 in MKS cells was almost undetectable. Scale bar, 5 µm. (B) Centrosomal localization of CEP290 (green) in control and mutant fibroblasts. The gamma-tubulin (γ-tub; red) labeling is used as a centrosomal marker. Image scale bar, 5 µm. Inset scale bar, 2 µm. (C) Quantification of the CEP290 immunofluorescence intensity at the basal body in each cell line. Values are the mean±SEM. Immunolabeling was performed from 90 to 100% confluent cells in two independent experiments. Automatic intensity measures were recorded in four to five fields. C corresponds to C1 and C2 pooled values. ****P<0.0001; n.s., not significant; A.U., arbitrary unit. Figure 5. View largeDownload slide CEP290 expression in quiescent cells. (A) Representative images of CEP290 (green) localization in control and mutant fibroblasts induced to quiescence. Acetylated α-tubulin (Ac-tub; red) is used to mark the ciliary axoneme. As in control cell lines (C1 and C2), CEP290 is correctly localized at the base of the cilia in patient (P1 and P2) and LCA10 (LCA10_1 and LCA10_2) fibroblasts. CEP290 in MKS cells was almost undetectable. Scale bar, 5 µm. (B) Centrosomal localization of CEP290 (green) in control and mutant fibroblasts. The gamma-tubulin (γ-tub; red) labeling is used as a centrosomal marker. Image scale bar, 5 µm. Inset scale bar, 2 µm. (C) Quantification of the CEP290 immunofluorescence intensity at the basal body in each cell line. Values are the mean±SEM. Immunolabeling was performed from 90 to 100% confluent cells in two independent experiments. Automatic intensity measures were recorded in four to five fields. C corresponds to C1 and C2 pooled values. ****P<0.0001; n.s., not significant; A.U., arbitrary unit. The CEP290 protein produced in P1 and P2 cells are able to incorporate into a macromolecular complex CEP290 has been reported to contribute to a macromolecule as large as 2–3 MDa (10). To determine whether the CEP290 protein produced in P1 and P2 cells retained this ability, protein extracts were analyzed by BN-PAGE using antibodies specific to CEP290 and Vinculin (loading control). A purified mitochondrial membrane protein extract was loaded onto the gel to use 1 MDa mitochondrial complex 1 (revealed by an anti-GRIM19 antibody) as a high molecular weight protein marker. Consistent with western blot analysis, the CEP290 antibody revealed no complex in the MKS protein extract (Fig. 6). In all other cell lines, we observed a macromolecular complex exceeding 1 MDa (Fig. 6). We conclude that the loss of amino acid residues encoded by Exons 18 and 8 and/or 32 does not significantly impair the capability of CEP290 to interact with some or all its partners in the complex (Fig. 3). Figure 6. View largeDownload slide Assembly of CEP290 in a high-molecular weight protein complex. Blue native-polyacrylamide gel electrophoresis analysis of protein extracts from control (C1 and C2) and mutant (P1, P2, LCA10_1, LCA10_2 and MKS) fibroblasts. Immunodetection using a CEP290 antibody reveals the presence of a high molecular weight complex in all cells lines but the MKS. Vinculin complex detection serves as a loading control. OXPHOS proteins from isolated mitochondrial membranes (Mt extract) were loaded and complex 1 of the respiratory chain (Mt Complex I) was immunolabeled using an anti-GRIM19 antibody to serve as the high molecular weight (1 MDa) marker. Figure 6. View largeDownload slide Assembly of CEP290 in a high-molecular weight protein complex. Blue native-polyacrylamide gel electrophoresis analysis of protein extracts from control (C1 and C2) and mutant (P1, P2, LCA10_1, LCA10_2 and MKS) fibroblasts. Immunodetection using a CEP290 antibody reveals the presence of a high molecular weight complex in all cells lines but the MKS. Vinculin complex detection serves as a loading control. OXPHOS proteins from isolated mitochondrial membranes (Mt extract) were loaded and complex 1 of the respiratory chain (Mt Complex I) was immunolabeled using an anti-GRIM19 antibody to serve as the high molecular weight (1 MDa) marker. The functional network of interactions between CEP290, member RAS oncogene family, centrosomal protein of 110 kDa and pericentriolar material 1 and cilia formation is apparently unaltered in P1 and P2 fibroblasts CEP290 is localized at the centrosome through its interactions with pericentriolar material 1 (PCM1), member RAS oncogene family (RAB8A) and centrosomal protein of 110 kDa (CP110). During the transition of the cells from proliferation to quiescence, the primary cilia formation-suppressor CP110 is released. This leads to the disinhibition of CEP290 and RAB8A to promote ciliation and the relocalization of PCM1 in the pericentriolar zone (10–12). We examined the localization of these proteins by immunocytochemistry in quiescent fibroblasts. Consistent with the near-absence of CEP290 in the MKS cells, we observed no RAB8A signals in the few cells able to build a cilium (Fig. 7A and B), as well as PCM1 and CP110 concentric centrosomal accumulation (Fig. 7C–F). Ciliation was severely altered with very few cells having the ability to produce a cilium, the length of which was substantially abnormal (Fig. 8). In contrast, in P1 and P2 fibroblasts, similar to in LCA10_1 and LCA10_2 cells expressing some WT CEP290, RAB8A and CP110 signals were comparable to that in the controls (Fig. 7A–D). Considering PCM1, scatter dot plot representations show a compact distribution of signal intensities at centrosomes in P1 and P2 as in control cells. However, mean signal intensities were slightly increased in patient lines, suggesting a marginal accumulation at centrosomes (Fig. 7E and F). In contrast, in LCA10_1 and LCA10_2 cells (like in the MKS line though to a lesser extent), the distribution of PCM1 signal intensities was rather dispersed, with significantly higher mean signal intensities compared with the controls. This observation suggests that PCM1 accumulates at the centrosomes of most LCA10_1 and LCA10_2 cells (Fig. 7E and F). Interestingly, ciliation was apparently unaltered in P1 and P2, but not in LCA10_1 and LCA10_2, which have decreased cilia abundance and shorter axonemes (Fig. 8), suggesting a possible correlation between PCM1 redistribution and ciliation alterations. Figure 7. View largeDownload slide Localization and abundance of CEP290 centriolar satellite partners. (A) Representative images of RAB8A (red) localization in the cilia from control (C1 and C2) and mutant (P1, P2, LCA10_1, LCA10_2 and MKS) fibroblasts induced to quiescence. ARL13B (green) labeling is used to mark the ciliary membrane. Image scale bar, 5 µm. Inset scale bar, 5 µm. (B) Quantification of RAB8A-positive cilia. Values are the mean±SEM (n>80; two independent experiments). C corresponds to C1 and C2 pooled values. ***P<0.001, ****P<0.0001; n.s., not significant; A.U., Arbitrary unit. Representative images of (C) CP110 or (E) PCM1 (green) centrosomal staining in quiescent control and mutant fibroblasts. Centrosomes are labeled by gamma-tubulin (γ-tub.; red). Image scale bar, 5 µm. Inset scale bar, 2 µm. Quantification of (D) CP110 or (F) PCM1 immunofluorescence intensity at centrosomes in quiescent fibroblasts. Values are the mean±SEM. Both immunolabelings were performed from 90 to 100% confluent cells in two independent experiments. Automatic intensity measures were recorded from 4, 5 and 8 fields, for controls (C1 and C2), patients (P1, P2, LCA10_1 and LCA10_2) and MKS cells, respectively. C corresponds to C1 and C2 pooled values. ** P<0.01, ***P<0.001, ****P<0.0001; n.s., not significant; A.U., Arbitrary unit. Figure 7. View largeDownload slide Localization and abundance of CEP290 centriolar satellite partners. (A) Representative images of RAB8A (red) localization in the cilia from control (C1 and C2) and mutant (P1, P2, LCA10_1, LCA10_2 and MKS) fibroblasts induced to quiescence. ARL13B (green) labeling is used to mark the ciliary membrane. Image scale bar, 5 µm. Inset scale bar, 5 µm. (B) Quantification of RAB8A-positive cilia. Values are the mean±SEM (n>80; two independent experiments). C corresponds to C1 and C2 pooled values. ***P<0.001, ****P<0.0001; n.s., not significant; A.U., Arbitrary unit. Representative images of (C) CP110 or (E) PCM1 (green) centrosomal staining in quiescent control and mutant fibroblasts. Centrosomes are labeled by gamma-tubulin (γ-tub.; red). Image scale bar, 5 µm. Inset scale bar, 2 µm. Quantification of (D) CP110 or (F) PCM1 immunofluorescence intensity at centrosomes in quiescent fibroblasts. Values are the mean±SEM. Both immunolabelings were performed from 90 to 100% confluent cells in two independent experiments. Automatic intensity measures were recorded from 4, 5 and 8 fields, for controls (C1 and C2), patients (P1, P2, LCA10_1 and LCA10_2) and MKS cells, respectively. C corresponds to C1 and C2 pooled values. ** P<0.01, ***P<0.001, ****P<0.0001; n.s., not significant; A.U., Arbitrary unit. Figure 8. View largeDownload slide Size and abundance of cilia. (A) Representative images of cilia in the quiescent control (C1 and C2) and mutant (P1, P2, LCA10_1, LCA10_2 and MKS) fibroblasts. The cilium axoneme is stained with acetylated α-tubulin (Ac-tub; red) and the basal body is marked with pericentrin (PCN; green). Scale bar, 5 µm. (B, C) Ciliogenesis analysis. (B) Abundance of ciliated cells and (C) length of cilia axonemes in control and mutant fibroblasts. Values are the mean±SEM. A minimum of 120 ciliated cells from three biological replicates were considered for each cell lines. C corresponds to C1 and C2 pooled values. *P<0.05, **P<0.01, ****P<0.0001, n.s., not significant. Figure 8. View largeDownload slide Size and abundance of cilia. (A) Representative images of cilia in the quiescent control (C1 and C2) and mutant (P1, P2, LCA10_1, LCA10_2 and MKS) fibroblasts. The cilium axoneme is stained with acetylated α-tubulin (Ac-tub; red) and the basal body is marked with pericentrin (PCN; green). Scale bar, 5 µm. (B, C) Ciliogenesis analysis. (B) Abundance of ciliated cells and (C) length of cilia axonemes in control and mutant fibroblasts. Values are the mean±SEM. A minimum of 120 ciliated cells from three biological replicates were considered for each cell lines. C corresponds to C1 and C2 pooled values. *P<0.05, **P<0.01, ****P<0.0001, n.s., not significant. The trafficking of retinitis pigmentosa GTPase regulator, intraflagellar transport 25 and intraflagellar transport 88 is apparently normal in P1 and P2 fibroblasts To assess cilia trafficking in cells expressing CEP290 proteins lacking amino acid sequences encoded by Exons 18 and 8 and/or 32, we analyzed the subcellular localization of retinitis pigmentosa GTPase regulator (RPGR), intraflagellar transport (IFT) 25 and IFT88 by immunocytochemistry in quiescent mutant and control cells. Consistent with unaltered cilia trafficking, the subcellular localization of RPGR, IFT25 and IFT88 in P1 and P2 cell lines was comparable to that of LCA10_1, LCA10_2 and control individuals (Fig. 9). Figure 9. View largeDownload slide Intraflagellar trafficking. Representative images of (A) RPGR, (C) IFT25 or (E) IFT88 (green) localization in the cilium in quiescent control (C1 and C2) and mutant (P1, P2, LCA10_1, LCA10_2 and MKS) fibroblasts. The ciliary axoneme is marked by the glutamylated-tubulin (GT-335; red) or acetylated α-tubulin (Ac-tub.; red). Scale bar, 5 µm. Quantification of (B) RPGR-, (D) IFT25- or (F) IFT88-positive cilia. Values are the mean±SEM (n>80 for each condition; two independent experiments). C corresponds to C1 and C2 pooled values. ***P<0.001, ****P<0.0001, n.s., not significant. Figure 9. View largeDownload slide Intraflagellar trafficking. Representative images of (A) RPGR, (C) IFT25 or (E) IFT88 (green) localization in the cilium in quiescent control (C1 and C2) and mutant (P1, P2, LCA10_1, LCA10_2 and MKS) fibroblasts. The ciliary axoneme is marked by the glutamylated-tubulin (GT-335; red) or acetylated α-tubulin (Ac-tub.; red). Scale bar, 5 µm. Quantification of (B) RPGR-, (D) IFT25- or (F) IFT88-positive cilia. Values are the mean±SEM (n>80 for each condition; two independent experiments). C corresponds to C1 and C2 pooled values. ***P<0.001, ****P<0.0001, n.s., not significant. Discussion Here, we report on the identification of homozygosity and compound heterozygosity for CEP290 mutations predicted to truncate the protein in two unrelated adult individuals presenting with early-onset and severe retinal dystrophy but notable preservation of central vision over two to three decades. This presentation contrasts with the typical ophthalmologic phenotype described in individuals carrying biallelic CEP290 truncating mutations who present a congenital and dramatically severe retinal dystrophy with major central dysfunction, which is inconsistent with useful vision (3,7). Consistent with the predictive model of CEP290 disease pathogenesis, which suggests that the phenotype is correlated with the amount of full-length or minimally shortened functional protein that can be produced from mutant alleles (3), we observed that CEP290 proteins were produced in dermal cells from P1 and P2, despite biallelism for presumably truncating mutations. mRNA analysis suggests that these proteins arose from differential splicing of Exons 18, 8 and 32, producing minimally shortened mRNA isoforms with intact reading frames escaping NMD, contrary to full-length mutant mRNA. Previously, Drivas et al. reported that CEP290 Exons 6, 10, 41 and 46 undergo non-canonical BES in WT skin fibroblasts (3). Here, studying the same cell-type, we detected CEP290 isoforms lacking either Exon 18 or Exon 32 but not Exon 8. BES of Exon 18 is probably the primary mechanism that enables the production of a CEP290 protein in P1 cells by providing them with the ability to accumulate a minimally shortened isoform that has an intact reading frame. In addition to setting Exons 17 and 19 in the same frame, the c.1666del mutation may also contribute conferring a lower chance of skipping mutant Exon 17 than the WT. This could synergize with the skipping of the adjacent PTC-encoding Exon 18. Recently, pre-mRNA splicing of CEP290 in iPSC-derived optic cups has been shown to differ significantly from that observed in fibroblasts (13), questioning the relevance of spontaneous skipping of Exon 18 in dermal cells with respect to the retina. Interestingly, analyzing a WT human fetal retina, we detected CEP290 lacking Exon 18, the amount of which was significantly higher than its counterpart in fibroblasts, as was that of the full-length isoform. This PTC-containing isoform likely undergoes NMD as it does in dermal cells (Fig. 2A, D and G). Thus the level of Exon 18 BES in the target tissue cannot be known. Likewise, in the absence of P1 iPSC-derived optic cups, the amount of PTC-free CEP290 lacking Exon 18 cannot be determined. In contrast to the situation observed in P1 cells, BES could not account alone for the skipping of Exon 32 in fibroblasts from P2, which occurred at a far higher level than in controls. ESE sequences are usually purine- or A/C-rich (14,15) and reduced enhancer activity has been reported when a T is introduced into an artificial polypurine sequence, mimicking an ESE of the dystrophin gene, where the suppression of the enhancer is more pronounced when a T creates a nonsense codon compared with a missense codon (16). Interestingly, the c.4090G>T mutation in Exon 32 introduces a T in a purine-rich sequence, predicting an alteration of an ESE with increased chance of exon skipping, whereas the introduction of any other nucleotide at this position does not (Table 2). This suggests that the c.4090G>T variation participates in the significant elevation of the amount of CEP290 lacking Exon 32 in P2 cells compared with controls through a mechanism known as Class I NAS. This type of NAS, triggered by ESE disruption, contrasts with Class II NAS, which is elicited by the disruption of the reading-frame through a nuclear scanning mechanism that recognizes the reading frame of the pre-mRNA (17). Regarding Exon 8, the c.508A>T mutation, like the c.4090G>T variation in Exon 32, introduces a T in a purine-rich ESE sequence and increases the probability of exon skipping, whereas any other nucleotide change at this position does not (Table 2). Considering that we observed CEP290 lacking Exon 8 in P2 fibroblasts but not in controls and WT retina, it is likely that the skipping is owing to Class I NAS, contrasting with that of Exons 18 and 32 involving BES and BES with NAS, respectively. CEP290 is one among many proteins that localize at the centrosome, the main microtubule-organizing center in eukaryotes. While centrosomes participate in the cell cycle, upon the transition from proliferation to quiescence, they migrate toward the cell surface to initiate cilia formation (18). CEP290 is among the most important proteins in this process. It is a regulator of cilia formation through its interaction with PCM1, RAB8A and CP110, which are the major components of a functional network whose integrity is required for proper ciliation. CEP290 localizes to centriolar satellites and binds to PCM1, where it recruits RAB8A (11). CP110 binds to and antagonizes CEP290. Upon entry into quiescence, CP110 is released, allowing ciliation (10). CEP290 depletion has been reported to alter the centrosomal distribution of PCM1 (accumulation) and RAB8A (abrogation), leading to cilia formation defects. Depletion of CEP290 had no effect on CP110 localization and level, as determined by immunofluorescence and western blot analysis, respectively (10). In MKS cells where no CEP290 could be detected upon immunoblot analyzes, the PCM1 and RAB8A distribution was altered as expected and we observed a highly significant accumulation of CP110 at the centrosome as determined by quantitative immunofluorescence analysis (Fig. 7). In homozygous P1 cells, the abundance of the truncated CEP290 produced from the mRNA lacking Exon 18 was ∼10% that of the WT in controls (Fig. 4), whereas in compound heterozygous P2 cells, the amount of CEP290 isoforms produced from both skipped mRNA species detected by RT-PCR (i.e. lacking Exon 8 or 32) was significantly higher (30% of the WT in controls; P < 0.05; Fig. 4). The amount of a high molecular weight complex involving CEP290 was also likely reduced in P1 compared with P2 cells (Fig. 6). Both observations could be owing to differential expression of skipped mRNA in P1 and P2 cells, but this hypothesis cannot be verified since RT-qPCR does not allow comparison of different mRNA species in two or more samples. Alternatively, the CEP290 protein expressed in P1 cells lacking 38 residues of the central homo/heterodimerization domain encoded by Exon 18 could have reduced stability compared with the isoforms deleted of short regions of the NH2-terminal homo/heterodimerization and RAB8A interaction domains encoded by Exons 8 and 32, respectively (Fig. 3). In contrast, whether CEP290 in P1 cells has reduced interaction abilities is unlikely. If it does, this would probably alter the amount of CEP290-interacting proteins at centrosomes, as seen in P2 cells where moderate reduction in RAB8A staining is observed which could be reasonably attributed to the altered binding properties of the CEP290 isoform lacking residues encoded by Exon 32. Very interestingly, despite significantly reduced and variable levels of shortened CEP290 isoforms as determined by immunoblot analysis, the amount of CEP290 measured by immunofluorescence at the centrosomes was similar in P1 and P2 cells, reaching ∼50% of the WT and allowing normal ciliation. Together these observations support the view that (i) truncated CEP290 isoforms accumulate at the centrosome upon serum starvation, (ii) these isoforms conserve efficient interactions with key centrosomal proteins, including RAB8A whose minor depletion has no obvious effect on ciliation (Figs 5 and 7). These observations also suggest that the c.1666del, c.508A>T and c.4090G>T mutations could be regarded as hypomorphic variants. Interestingly, while to our knowledge homozygosity for the c.1666del has not been described previously, the change has been reported to cause Joubert syndrome (JS) in association with the c.3904C>T (p.Gln1302*), c.6031C>T (p.Arg2011*) or c.6012–12T>A mutations that affect Exons 31, 44 and intron 43, respectively (19–21). The c.6012–12T>A mutation abolishes the role of the natural acceptor splice site of intron 43 by activating a stronger neighboring cryptic splice site, the use of which introduces 56 nucleotides of the intronic sequence and a PTC in the mRNA. The c.3904C>T and c.6031C>T nonsense mutations confer to Exons 31 and 44 a higher chance of skipping compared with WT (Supplementary Material, Table S3). While the skipping of Exon 44 would disrupt the reading frame, that of Exon 31 would allow bypassing PTC. However, the encoded protein would lack a large stretch of 150 amino acids that are involved in RAB8A binding, presumably disrupting the RAB8A, PCM1 and CP110 functional complex involved in cilia formation (11). Hence, alleles found in combination with the c.1666del in JS are unlikely to encode functional CEP290, in accordance with the CEP290 predictive model of disease pathogenesis. Regarding Exon 32, unlike Exon 8, it has been involved in other individuals suffering from ciliopathies. The c.4090G>T change has been described in association with the c.3265C>T nonsense mutation (p.Gln1089*) in a LCA10 individual (22). This other nonsense mutation lies within Exon 28 that, if skipped, would produce another PTC, hence hampering the ability of cells in the individual to escape protein truncation. The c.4115–4116del introduces a PTC within Exon 32 (p.Ile1372Lysfs*5). Unlike the c.4090G>T mutation, it is predicted to reduce the chance of skipping Exon 32, thus producing no CEP290. The identification of this mutation in a LCA10 individual harboring the hypomorphic c.2991+1655A>G substitution and in an MKS fetus carrying the c.1219–1220del (p.Met407Glufs*14) in Exon 14, for which skipping would disrupt the reading frame, is consistent with this view. Intriguingly, while a low amount of minimally shortened CEP290 allowed apparently unaltered ciliation in the P1 and P2 cells, marked PCM1 redistribution and cilia defects were noted in LCA10_1 and LCA10_2, which expressed WT CEP290 (Figs 7E, F and 8). Of note cilia anomalies in LCA10_1 and LCA10_2 fibroblasts were less marked than the ones we reported earlier in the same cell lines [30% versus 50% of cells with absent cilium in (5)]. This variability might be essentially owing to the fact that we increased cell confluence in the present study [90–100% versus 80% in (5)]. Indeed, it has been reported that contact inhibition can induce quiescence and cilia formation even before serum-starvation (23). Extended serum-starvation in the present study [48 h versus 30 h in (5)] might also have influenced the kinetics of cilia formation. PCM1 redistribution and cilia formation alterations in LCA10_1 and LCA10_2 individuals raise the question of whether the c.2991+1655A>G allele produces a stable truncated protein that could interfere with the WT counterpart. The protein encoded by this allele would encompass the PCM1—but not the RAB8A—interacting domains. While CEP290 depletion has been reported to induce centrosomal PCM1 retention, the production of smaller size PCM1 complexes from the truncated protein could aggravate the phenomenon by altering the PCM1-dependent shuttling between the centrosome and the cytosol (11). Considering the ciliation anomalies, it is worth remembering that cellular expression of CEP290 fragments lacking the RAB8A domain of interaction have been shown to alter cilia formation (10). In summary, we report here naturally occurring exon skipping that produces CEP290 isoforms retaining functional interactions with centrosomal proteins important to cilia formation and intraflagellar trafficking, as the likely cause of unusually mild retinal disease in two unrelated individuals. While NAS has been previously suggested to account for atypical CEP290-related retinal diseases (8,9), our data suggest that BES can also contribute. Whatever the mechanism, the evidence of diminished severity in individuals producing low levels of minimally shortened protein supports the view that some CEP290 exons may be dispensable. This observation provides strong support of splice switching oligonucleotide-mediated therapies as a means to bypass protein truncation in patients with CEP290 mutations that introduce PTCs. Materials and Methods Patients Patient 1 (II: 2, Family 1), an 18-year-old woman, is the second of two children born to apparently unrelated parents of French origin. At the age of 18 months, she manifested uncomfortable mobility in dim light environments. Upon examination, she presented with hypermetropia (+4.5 and +5 diopters, right and left eyes, respectively), peripheral scotoma and extinct electroretinogram (ERG). Ophthalmoscopic examination was initially unremarkable, but retinal lesions were overt before teenage with multiple mid-peripheral white dots and cystoid macular edema at the fundus, cystic structures and severe thinning of the photoreceptor layer at optic coherence tomography and increased autofluorescence in the perimacular and inferior vascular arcade at autofluorescence imaging approximately 10, and peripheral osteoblast-like pigmentary deposits at 13 years of age, respectively. At the age of 18 years, the visual field was reduced to the central 10°, with some preserved peripheral islets and visual acuities of 20/40 and 20/30 (right and left eyes, respectively) (Table 1). The patient has received 200 mg acetazolamide daily since the age of 10 years old to treat her macular edema. Panel-based molecular diagnosis testing for inherited retinal diseases identified homozygosity for the CEP290 c.1666del (p.Ile556Phefs*17) mutation (Fig. 1A; Table 1). Patient 2 (II: 1, Family 2), a 33-year-old man, is the first of two children born to unrelated Portuguese parents. He presented at a genetic consultation with the Genetic Department of our hospital with a history of night blindness around the age of 2 years and a diagnosis of retinitis pigmentosa at the age of 7 years. Upon examination at the Ophthalmologic Department of our hospital at the age of 21 years, he presented with night blindness, a tubular visual field, fixation nystagmus, pure blue-yellow dyschromatopsia and visual acuities of 20/100 and 20/400 (right and left eyes, respectively). Fundoscopy showed absent macular reflex without rearrangement, dull retina without pigment migration at the fundus and thin retinal vessels. Examination at 31-year-old demonstrated no deterioration of the fundus aspect or of his visual acuities. The RD panel gene screening identified compound heterozygous CEP290 nonsense mutations c.508A>T (p.Lys170*) and c.4090G>T (p.Glu1364*) (Fig. 1B;Table 1). Familial analysis demonstrated biallelism and segregation of the mutations with disease in the two families (Fig. 1A and B). We also included in the study two individuals affected with LCA homozygously carrying the c.2991+1655A>G mutation (LCA10_1 and LCA10_2) and a fetus interrupted for MKS carrying the p.Arg205* change in homozygosity (MKS). These subjects were previously referred to as P1 and P2 (5), and Family 1_Subject 5 (24), respectively (Table 1). Written informed consent was obtained from all participating individuals or their legal representatives and the study was approved by the Comité de Protection des Personnes ‘Ile-De-France II’. Methods In silico analysis of mutations on splicing The effect on the splicing of the CEP290 mutations identified in the P1 and P2 patients was analyzed in silico using splice signal detection software {Human Splicing Finder, NNSPLICE from fruitfly.org, SpliceSiteFinder-like, MaxEntScan, GeneSplicer [Alamut Interpretation Software 2.0 (gateway for Human Splicing Finder, NNSPLICE, SpliceSiteFinder-like, MaxEntScan, GeneSplicer, ESEFinder, RESCUE-ESE and EX-SKIP), http://www.interactive-biosoftware.com; date last accessed May 22, 2018]}, ESE binding site detection software (ESEFinder, RESCUE-ESE), the EX-SKIP program, which compares the ESS/ESE profile of the WT, and a mutated allele to determine which exonic variant has the highest chance to skip this exon, and HOT-SKIP (HOT-SKIP program, http://hot-skip.img.cas.cz; date last accessed May 22, 2018), which systematically examines all possible substitutions in each exonic position that are most likely to skip the submitted exonic sequence. Primary cell culture Skin biopsies were obtained from the affected subjects: P1 (II: 2, Family 1), P2 (II: 1, Family 2), LCA10_1, LCA10_2, MKS and two control individuals (C1, C2) (Table 1). Primary fibroblasts were isolated by selective trypsinization and proliferated at 37°C with 5% CO2 in Opti-MEM Glutamax I medium (Life Technologies, Saint Aubin, France) supplemented with 10% fetal bovine serum (Life Technologies), 1% Ultroser-G serum substitute (Pall, Saint-Germain-en-Laye, France), 50 U/ml penicillin and 50 μg/ml streptomycin (Life Technologies). Cells were grown in 75 cm2 flasks (<12 passages) and cultured on a 0.1 mg/ml collagen support for fibroblasts from the MKS fetus. For the inhibition of nonsense-mediated mRNA decay, cells were seeded in a six-well plate before being incubated with 25 μg/ml of emetine dihydrochloride hydrate (Sigma-Aldrich, Saint-Quentin Fallavier, France) directly added to the fresh culture media for 15 h. RNA preparation and cDNA synthesis Total RNA from fibroblasts and WT human fetal (22 weeks) retina was extracted using the RNeasy Mini Kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s protocol. All samples were DNase-treated by the RNase-free DNase set (Qiagen). The concentration and purity of total RNA was determined using the Nanodrop-2000 spectrophotometer (ThermoScientific, Illkirch, France) before storage at −80°C. First-stranded cDNA synthesis was performed from 500 ng of total RNA extracted using the Verso cDNA kit (ThermoScientific) with random hexamer: anchored oligo(dT) primers at a 3:1 (vol:vol) ratio according to the manufacturer’s instructions. A non-reverse transcription reaction (without enzyme; RT-) for one sample was prepared to serve as a control for reverse transcription PCR (RT-PCR) and real-time quantitative PCR (RT-qPCR) experiments. RT-PCR CEP290 splicing isoforms were amplified from reverse transcribed mRNAs (2 µl) in 20 µl of 1× Phusion HF buffer containing 4 mM dNTPs (ThermoScientific), 0.4 units of Phusion High-Fidelity DNA polymerase (ThermoScientific) and 10 µM of specific primer pairs (Supplementary Material, Fig. S2 and Table S1). No template (NTC) reactions were used as negative controls. PCRs were carried out on a 2720 Thermal Cycler (Applied Biosystems, Courtaboeuf, France) under the following conditions: initial denaturation at 98°C for 5 min, followed by 32 cycles of 20 s denaturation at 98°C, 15 s annealing at 62°C and 30 s extension at 72°C. PCR products (10 µl) were separated by electrophoresis in a 3% low-melting agarose gel stained with ethidium bromide, visualized under UV lights and cut off. In-gel PCR products (2–5 μl; 65°C) were further sequenced using the Big Dye TerminatorCycle Sequencing Kit v3.1 (ABI Prism™, Applied Biosystems, Foster City, USA) on a 3500 automated sequencer (Applied Biosystems). RT-qPCR analysis The abundance of CEP290 mRNA isoforms was measured using primers specific to the unskipped (referred to as ‘full length’) or skipped CEP290 versions. Primer sequences are listed in Supplementary Material, Figure S2 and Table S2. GUSB (NM_000181.3), RPLP0 (NM_001002.3) mRNAs and the ALB (NM_000477) gene were used to normalize the data and control the non-contamination of cDNAs by genomic DNA, respectively (5). The cDNA (5 μl of a solution diluted at 1:25 in RNAse-free H2O) of each sample was subjected to PCR amplification in real-time in a buffer (20 μl) containing SYBR GREEN PCR Master Mix (Life Technologies) and 300 nM forward and reverse primers in the following conditions: activation of Taq polymerase and denaturation at 95°C for 10 min followed by 50 cycles of 15 s at 95°C, and 1 min at 60°C. The specificity of the amplified products was determined after the analysis of the melting curve carried out at the end of each amplification using one cycle at 95°C for 15 s, then a graded thermal increase of 60°C to 95°C for 20 min. The data analysis and methodology were performed as previously described (5). Western blot and blue native-polyacrylamide gel electrophoresis (BN-PAGE) analysis Cells were lysed on ice for 1 h by repeated homogenization in a low detergent lysis buffer containing phosphate buffered saline (PBS) 1×, 1% Triton, Halt™ Protease Inhibitor Cocktail 1X (ThermoScientific) and 25 U/ml Pierce Universal Nuclease (ThermoScientific). The lysates were centrifuged (20 000g at 4°C for 15 min), supernatants were collected and proteins quantified using the Bradford method. For western blot analysis, proteins (100 µg) were resolved by a 4–15% polyacrylamide gel (mini-PROTEAN TGX, Bio-Rad, Marnes-la-Coquette, France) according to the supplier’s recommendations. All lysates were heated at 95°C for 10 min prior to loading. Proteins were transferred to a PVDF 0.2 µM membrane (Bio-Rad) using a Trans-Blot Turbo Transfer System (Bio-Rad), and then processed for immunoblotting. Membranes were probed with polyclonal rabbit anti-C-terminal-CEP290 (1:1500, Novus Biologicals, USA) and monoclonal mouse anti-β-actin (1:2000, Abcam, Paris, France) primary antibodies, and then incubated with donkey anti-rabbit IgG-HRP (1:2000, ThermoScientific) and donkey anti-mouse IgG-HRP secondary antibodies (1:4000, ThermoScientific), respectively. Blots were developed with the use of the Clarity Western ECL Substrate (Bio-Rad) and ChemiDoc XRS+ Imaging System (Bio-Rad). Western blot images were acquired and analyzed with Image Lab software 3.0.1 build 18 (Bio-Rad). The abundance of CEP290 relative to β-actin was estimated in each cell line by densitometry using Image Lab software (Bio-Rad). CEP290 abundance levels are the means of five independent protein extracts. For BN-PAGE, the proteins (100 µg) were solubilized by 250 U/µl Pierce Universal Nuclease (ThermoScientific) and loaded on a native 4–16% Bis-Tris gel (Life Technologies). Native proteins were transferred to a PVDF membrane (Merck-Millipore, Fontenay sous Bois, France) overnight at 25 V and 4°C in transfer buffer containing 0.02% SDS, and processed for immunoblotting as described above. Protein complexes containing Vinculin were immunolabeled by rabbit anti-Vinculin antibody (1:10 000; Abcam) to serve as the loading control. OXPHOS proteins from isolated mitochondrial membranes (20 µg) were solubilized by 2% lauryl maltoside (25), were loaded and complex 1 of the respiratory chain was immunolabeled using an anti-GRIM19 antibody (1:10 000; Abcam) to serve as the high molecular weight (1 MDa) marker. Immunocytochemistry analysis Cells were grown on glass coverslips in a 12-well plate before being incubated for 48 h in serum-free medium. After serum-starvation, the cells were fixed with cold methanol (7 min at –20°C) and washed two times with PBS. Cells were permeabilized and nonspecific sites were saturated in a PBS solution containing 5% normal goat serum, 3% bovine serum albumin and 0.5% Triton X-100 for 1 h. Permeabilized cells were incubated overnight at 4°C with primary antibodies in PBS containing 3% bovine serum albumin and 0.1% Triton X-100:anti-Pericentrin rabbit antibody (1:1000, Abcam), anti-γ-tubulin mouse antibody (1:500, Sigma-Aldrich), anti-CEP290 rabbit antibody (1:100, Novus Biologicals), anti-CP110 rabbit antibody (1:100, ProteinTech), anti-PCM1 rabbit antibody (1:100, Cell Signaling Technology), anti-acetylated α-Tubulin mouse antibody (1:1000, Sigma-Aldrich), anti-GT335 mouse antibody (1:100, Adipogen), anti-ARL13B rabbit antibody (1:100, ProteinTech), anti-IFT25 rabbit antibody (1:100, ThermoScientific) anti-IFT88 rabbit antibody (1:100, ProteinTech), anti-RPGR rabbit antibody (1:100, Sigma-Aldrich) and anti-RAB8A mouse antibody (1:50, Abnova). After three washes in PBS, the cells were incubated for 1 h at room temperature with secondary antibodies in PBS solution containing 3% bovine serum albumin and 0.1% Triton X-100:anti-rabbit IgG goat antibody coupled to Alexa-Fluor 488 and anti-mouse IgG goat antibody coupled to Alexa-Fluor 568 (1:1000, Life Technologies). After three additional washes with PBS, the coverslips were mounted on slides using a mounting medium containing DAPI (ProLong Gold antifade reagent with DAPI, Invitrogen) to stain the cell nuclei. Immunofluorescence images were obtained using a Zeiss LSM700 confocal microscope. Exposure times and settings for image processing were constant for all samples to allow sample comparison. The number of Z-stacks collected was variable between the samples but optimized for capturing maximum fluorescent signals. Deconvoluted images were projected into one picture using ImageJ software (https://imagej.nih.gov/ij; date last accessed May 22, 2018) Z-project tool with the maximum intensity setting. The total fluorescence contained in 7 and 16 µm2 squares centered on the centrosome as determined by γ-tubulin staining was recorded to measure the intensity of CEP290 and CP110 and that of the PCM1 labelling in the peri-centrosomal region, respectively. Integrated pixel densities were quantified in each square using ImageJ software and a default threshold for background subtraction of 30. The final images were generated using ImageJ software. Statistics The data obtained from C1 and C2 were systematically pooled. Prism6 software was used for statistical analyses. The significance of variation among samples was determined using one-way ANOVA with a post hoc Dunnett’s test in all experiments but RT-qPCR for which we used a post hoc Tukey’s test. Error bars reflect the standard error of the mean (SEM). Supplementary Material Supplementary Material is available at HMG online. Acknowledgements We gratefully acknowledge the patients that donated skin biopsies for this study. Conflict of Interest statement. None declared. Funding This work was supported by Retina France, by grants from the Fondation JED-Belgique to X.G., UNADEV-AVIESAN/ITMO NNP (R16074KS) to I.P. and UNADEV-AVIESAN/ITMO NNP (R16073KS) to J.-M.R. References 1 Ware S.M. , Aygun M.G. , Hildebrandt F. ( 2011 ) Spectrum of clinical diseases caused by disorders of primary cilia . Proc. Am. Thorac. Soc ., 8 , 444 – 450 . Google Scholar CrossRef Search ADS PubMed 2 Papon J.F. , Perrault I. , Coste A. , Louis B. , Gérard X. , Hanein S. , Fares-Taie L. , Gerber S. , Defoort-Dhellemmes S. , Vojtek A.M. et al. ( 2010 ) Abnormal respiratory cilia in non-syndromic Leber congenital amaurosis with CEP290 mutations . J. Med. Genet ., 47 , 829 – 834 . 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Human Molecular GeneticsOxford University Press

Published: May 16, 2018

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