Identification of Novel Variants in <i>LTBP2</i> and <i>PXDN</i> Using Whole-Exome Sequencing in Developmental and Congenital Glaucoma - Fig 3

(a) DNA chromatogram of the relevant PXDN fragment for the carrier and homozygous variant are shown (b). Family pedigree and segregation of a novel missense mutation (c.3496G>A; p.Gly1166Arg) in the PXDN gene. (c). Multiple sequence alignment of the region of the PXDN protein surrounding the novel Gly1166Arg mutation in various species. The glycine residue (indicated with an arrow) is highly conserved among all species analyzed.</p

Distribution of single nucleotide polymorphisms rs10490924 in <i>ARMS2</i> and rs1061170 in <i>CFH</i> and trend test of AMD severity stages (data of other SNPs is shown in S1 Table).

<p>Distribution of single nucleotide polymorphisms rs10490924 in <i>ARMS2</i> and rs1061170 in <i>CFH</i> and trend test of AMD severity stages (data of other SNPs is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156778#pone.0156778.s001" target="_blank">S1 Table</a>).</p

Identification of Novel Variants in <i>LTBP2</i> and <i>PXDN</i> Using Whole-Exome Sequencing in Developmental and Congenital Glaucoma - Fig 2

<p>(a) Sanger sequencing chromatograms for the carrier III:1 and affected individual IV:2 homozygous for the mutation (b) Family pedigree and segregation of a novel frameshift mutation (c.4031_4032insA; p.Asp1345Glyfs*6) in the <i>LTBP2</i> gene in a PCG family.</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

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

Univariate regression analysis for SNPs rs10490924 in ARMS2 and rs1061170 in CFH in patients with unilateral nAMD and different severity stages of the fellow-eye.

<p>Univariate regression analysis for SNPs rs10490924 in ARMS2 and rs1061170 in CFH in patients with unilateral nAMD and different severity stages of the fellow-eye.</p

Expression of the full-length N-SF-TAP-Rp1 and N-LAP-Rp1 proteins prevents photoreceptor degeneration in Rp1^Q662X/Q662X knock-in mice.

<p>A. Frozen sections of retina from 2-month-old mice of the genotypes indicated were stained with anti-C-Rp1 antibodies (red). The wild-type Rp1 protein is located in the axonemes of PSC; full-length Rp1 protein is not detected in the <i>Rp1</i>-Q662X knock-in mice. Full-length Rp1 protein is also detected in the PSC axonemes of the <i>Rp1</i>-Q662X : N-SF-TAP-<i>Rp1</i> and <i>Rp1</i>-Q662X : N-LAP-<i>Rp1</i> mice. There is a mosaic pattern of full-length Rp1 protein expression in the <i>Rp1</i>-Q662X : N-LAP-<i>Rp1</i> mice. (INL, inner nuclear layer; IS, inner segment; ONL, outer nuclear layer; OS, outer segment; 400X magnification for all images). B. Retinal histology from 2-month-old mice of the genotypes indicated. The retinal structure of the <i>Rp1</i>-Q662X : N-SF-TAP-<i>Rp1</i> mice is normal, in contrast to the early photoreceptor degeneration in the <i>Rp1</i>-Q662X mice. There is partial preservation of retina structure in the <i>Rp1</i>-Q662X : N-LAP-<i>Rp1</i> mice (INL, inner nuclear layer; IS, inner segment; ONL, outer nuclear layer; OS, outer segment; 400× magnification for all images). C. Ultrastructure of PSCs in two-month old mice of the genotypes indicated. Note that the structure of the PSC and organization of the outer segment discs are normal in the <i>Rp1</i>-Q662X : N-SF-TAP-<i>Rp1</i> mice, in contrast to the disorganized PSC observed in the <i>Rp1</i>^Q662X/Q662X mice. There is a mixture of cells with normal PSC and cells with disorganized PSC in the <i>Rp1</i>-Q662X : N-LAP-<i>Rp1</i> mice (OS, outer segment; RPE, retinal pigment epithelium. Bars  = 2 µm). D. Amplitudes of ERG responses from 2 month-old and 12-month-old mice of the genotypes indicated. At 2 months not that the rod and cone ERG amplitudes are close to normal in the <i>Rp1</i>-Q662X : N-SF-TAP-<i>Rp1</i> mice, in contrast to the reduction of photoreceptor function observed in the <i>Rp1</i>^Q662X/Q662X mice. There is partial restoration of photoreceptor function in the <i>Rp1</i>-Q662X : N-LAP-<i>Rp1</i> mice. At 12 months photoreceptor function is relatively well preserved in the <i>Rp1</i>-Q662X : N-SF-TAP-<i>Rp1</i> mice. There is also some residual photoreceptor function in the <i>Rp1</i>-Q662X : N-LAP-<i>Rp1</i> mice. Significant differences compared to controls are indicated by * (P<0.01) and ** (P<0.05).</p

Multivariate regression model for each severity stage with results for SNPs rs10490924 in <i>ARMS2</i> and rs1061170 in <i>CFH</i> (data of other SNPs is shown in S3 Table).

<p>Multivariate regression model for each severity stage with results for SNPs rs10490924 in <i>ARMS2</i> and rs1061170 in <i>CFH</i> (data of other SNPs is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156778#pone.0156778.s003" target="_blank">S3 Table</a>).</p

Grading results.

<p>Grading results.</p

Area under the curve (AUC) of risk models in different AMD severity stages.

<p>Area under the curve (AUC) of risk models in different AMD severity stages.</p