Summary of clinical findings.

Summary of clinical findings.</p

CEP290 protein analysis in <i>Cep290</i> mouse models.

<p>Representative CEP290 immunodetection in P150 retinas from the three different mouse models. A CEP290-Flag construct was expressed in HEK293-T cells and used as a positive control. Tubulin was used for normalization.</p

Generation of the <i>Cep290</i> humanized mouse models.

<p>(A) Structure of the <i>CEP290</i> gene in human and mouse (drawn to scale). (B) Two mouse models were generated by introducing human exons 26 and 27 and intron 26, either with or without the LCA-causing mutation (depicted with *), to the mouse <i>Cep290</i> gene. Human and mouse loci, targeting vector and recombinant locus are depicted. Arrows and letters indicate the position of the oligonucleotides (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079369#pone.0079369.s001" target="_blank">Table S1</a>). </p

Analysis of the <i>CEP290</i> cryptic exons.

<p>(A) Expected transcripts for each model. (B) Exon Y is expressed in all tissues in either <i>Cep290</i>^{<i>hum/hum</i>} and <i>Cep290</i>^{<i>lca/lca</i>} mice. (C) Only in the <i>Cep290</i>^{<i>lca/lca</i>} model, exon X is expressed, either with exon Y or without, at very low levels. The arrow indicates the band that contains both cryptic exons (exon X and exon Y) in the same transcript. <i>Cep290</i>^lca/lca PCR products were run longer to clearly separate the two different bands. (D) Schematic representation of the different transcripts containing cryptic exons found in the three mouse models. Mouse exons and introns are depicted in black, while human exons and introns are represented in blue. Cryptic exons X and Y are shown in grey and red, respectively. Arrows and letters indicate the position of the oligonucleotides in the cryptic exons. Semi-quantification was performed using ImageJ software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079369#B21" target="_blank">21</a>]. MQ: H₂O (negative control); F: human LCA fibroblasts; B: Brain; K: Kidney; R: Retina; Lu: Lung; S: Spleen; T: Testis and Li: Liver.</p

Putative pathogenic variants in 25 unrelated German patients initially diagnosed with LCA.

Putative pathogenic variants in 25 unrelated German patients initially diagnosed with LCA.</p

Assessment of pathogenicity of missense variants identified in this study.

Assessment of pathogenicity of missense variants identified in this study.</p

Morphological and CEP290 immunolocalization analyses in P150 mouse retinas.

<p>Immunodetection of CEP290 and acetylated tubulin in retinal sections from <i>Cep290</i>^<i>wt/wt</i>, <i>Cep290</i>^{<i>hum/hum</i>} and <i>Cep290</i>^{<i>lca/lca</i>} mice, at P150. Images were taken at 200X (A) and 400x (B) magnifications. CEP290 (green) is located in the connecting cilium (CC) and remains unaltered in the humanized models. Nuclei were marked with DAPI (blue) and CC with acetylated tubulin (red). GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; PhL: photoreceptor layer; RPE: retinal pigment epithelium; IS: inner segment; CC: connecting cilium; OS: outer segment.</p

Expression of N-SF-TAP-Rp1 and N-LAP-Rp1 Transgenes.

<p>A. Diagram of <i>Rp1</i> gene, N-SF-TAP-<i>Rp1</i> and N-LAP-<i>Rp1</i> transgenes. The TAP and LAP tags were introduced into the beginning of the <i>Rp1</i> coding sequence in exon 2 in BAC 314, which contains 140 kb of mouse genomic DNA surrounding the <i>Rp1</i> locus. B-C. Western blot analyses of Rp1 proteins in N-SF-TAP-<i>Rp1</i> and N-LAP-<i>Rp1</i> mice. Equal amounts of protein from retinal extracts of wild-type, N-SF-TAP-<i>Rp1</i> and N-LAP-<i>Rp1</i> mice were analyzed by Western blotting using anti-Rp1 antibodies. The blots were also probed with antibodies to ATPase as a loading control. The Rp1 levels for the different transgenic lines were quantified and normalized to the ATPase signals. B. N-SF-TAP-<i>Rp1</i> mice. The total level of Rp1 protein in line T1 was 144% of that observed in non-transgenic littermate controls, indicating that the transgene increased expression approximately 44%, or nearly the amount expected from a third <i>Rp1</i> allele. The N-SF-TAP-<i>Rp1</i> transgene in line T2 is over-expressed relative to the wild-type protein, as it increased the total Rp1 protein level to ∼300% of normal. The total level of Rp1 protein in line T3 was only slightly elevated, but the retinas in these mice were also significantly degenerated, with 40% of the photoreceptor nuclei remaining in the outer nuclear layer, suggesting that the N-SF-TAP-Rp1 protein in this transgenic line is also 2–3 fold greater than wild-type. C. N-LAP-<i>Rp1</i> mice. The levels of N-LAP-<i>Rp1</i> fusion protein in lines L1 and L2 mice were approximately half of that observed for the wild-type Rp1 protein, again indicating that the transgene increased expression approximately the amount expected from a third <i>Rp1</i> allele. In contrast, N-LAP-<i>Rp1</i> transgene in line L3 is over-expressed, and increased the total Rp1 protein level to ∼250% of normal. D. Immunofluorescence analyses of wild-type Rp1 protein (anti-Rp1 antibodies; red) and N-SF-TAP-Rp1 protein (anti-FLAG antibodies; green) in three N-SF-TAP-<i>Rp1</i> transgenic lines and wild-type littermate control. Note that the wild-type Rp1 protein is located in the axoneme of photoreceptor outer segments. The N-SF-TAP-Rp1 protein in transgene line T1 shares the same location, as indicated by the overlap of the two signals in the merged image panel (bottom left). There is also some N-SF-TAP-Rp1 signal in the synaptic region of photoreceptor cells that is not present in wild-type retinas. The N-SF-TAP-<i>Rp1</i> transgenes in T2 and T3 are over-expressed relative to the wild-type protein. The over-expressed N-SF-TAP-Rp1 protein localizes correctly to PSC axonemes, but also mis-localizes to photoreceptor inner segments. The N-SF-TAP-Rp1 signal in the synaptic region is also increased, especially in the line T3 retinas. Note that the outer nuclear layer is thinner in the line T3 sample, consistent with the photoreceptor degeneration observed in this transgene line. The ONL is also slightly thinner in the line T2 samples as well. E. Immunofluorescence analyses of wild-type Rp1 protein (anti-Rp1 antibodies; red) and N-LAP-Rp1 protein (EGFP; green) in three N-LAP-<i>Rp1</i> transgenic lines and wild-type littermate control. The N-LAP-Rp1 protein in transgene line L1 is located in the axoneme of PSCs, like the wild-type protein. In addition, there is EGFP signal from N-LAP-Rp1 protein in the inner segments and cell bodies of the photoreceptors. Since this was not detected by the anti-Rp1 antibodies, it must be due to truncated versions of the N-LAP-Rp1 protein that retain the N-terminal EGFP tag, but have lost the C-terminal antibody binding domain. The N-LAP-<i>Rp1</i> transgenes in line L3 is over-expressed relative to the wild-type protein. The over-expressed N-SF-TAP-Rp1 protein localizes correctly to PSC axonemes, but also mis-localizes to photoreceptor inner segments and cell bodies. As for the N-SF-TAP-Rp1 line T3, there is photoreceptor degeneration in the L3 line. The N-LAP-Rp1 transgene expression in line L2 is not completely uniform, with some cells that do not express the transgene evident. In addition, there are red signals in OPL in line L1 and L2, which could represent the non-specific signaling from the anti-c-Rp1 antibody. It is also possible that this immunoreactivity of Rp1 could correspond to the C-terminal fragments of Rp1. (IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment; 400X magnification for all images).</p

Transcriptional characterisation of <i>Cep290</i> in humanized mouse models.

<p>(A-B) <i>Cep290</i> expression levels in various mouse tissues were assessed by RT-PCR. Regions containing murine exons 10 to 13 (A) and 24 to 27 (B) were analyzed. No differences were observed. (C) Amplification using human primers was also assessed in the three models. Only humanized models showed amplification. (D) Actin was used for normalization and comparison among tissues and models. MQ: H₂O (negative control); B: Brain; K: Kidney; R: Retina; Lu: Lung; S: Spleen; T: Testis and Li: Liver.</p

<i>RP1</i> Gene, Clinical and Sequence Data for Family W04-348.

<p><b>A. </b><i>RP1</i> gene and identified mutations. The gene structure of <i>RP1</i> is depicted, with the locations of mutations that cause adRP and arRP indicated; the mutations above the <i>RP1</i> gene structure cause dominant RP, labeled as adRP in red; whereas mutations below the <i>RP1</i> gene structure cause recessive RP, labeled as arRP in blue; frameshift mutation p.P229QfsX35 reported to cause arRP in this study is in bold. The portion of the gene that encodes that DCX domains is also indicated. The arrow on the top indicates the location of the R677X (human) and Q662X (mouse) mutations. <b>B.</b> Pedigree for family W04-348. The c.686delC, p.P229QfsX35 mutation is designated by <i>M</i>. <b>C.</b> ERG traces from a normal control, the patient’s parents (I-1, I-2 (age 57), and the affected patient (II-1). The five standard ISCEV recordings are shown, from the top including: scotopic rod responses, scotopic combined rod-cone responses, oscillatory potentials, photopic single flash and photopic 30Hz responses. The amplitude and time scales are indicated. The ERG responses of the patient’s parents show normal amplitudes and implicit times; the patient had no recordable rod or cone responses. The deflections shown in the 30 Hz recordings for the patient are due to motion artifact. The thicker traces are from the right eye, the thinner from the left eye. <b>D.</b> Fundus photos (left) and fundus autofluorescence images (right) of the affected patient II-1(age 30) and his father I-1 (age 60). The patient has typical findings of RP, with optic disc pallor, attenuation of the retinal blood vessels, RPE atrophy and bone spicule pigmentation outside the macula. As shown in the autofluorescence image, the RPE in the macular region is relatively preserved. In contrast, the father’s fundi are normal. <b>E.</b> Sequence traces showing the homozygous c.686delC mutation in patient II-1, carrier status of this mutation in the patient’s father I-1, and the wild-type sequence in an unaffected Dutch control. Note that the sequence trace of the mutant allele in individual I-1 is shifted slightly.</p