Fundus photographs of the control and CHM patients.

<p><b>a.</b> CHM patient 22 y.o. characterized by RPE depigmentation and widespread RPE disruption <b>b.</b> CHM patient 74 y.o. characterized by loss of RPE and choroid, scattered pigment in macula, faint deep choroidal vessels and severely narrowed retinal vessels and optic nerve pallor (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008402#pone-0008402-t001" target="_blank"><b>Table 1</b></a>, CHM 9 and 10 respectively). <b>c.</b> Female CHM carrier, age 50 showing patchy RPE hypopigmentation without pigment dispersion and control subject. <b>d.</b> Fundus photograph of the normal eye.</p

Lysosomal acidification and rate of proteolytic degradation in monocytes from CHM and control patients treated with Bafilomycin-A1 (BafA1).

<p><b>a.</b> Lysosomal acidification and rate of proteolytic degradation in monocytes from CHM and control BafA1. Intralysosomal acidification measurements were performed using <i>E. coli</i> BioParticles conjugated with a pH dependent dye (pHrodo). Treatment caused an increase in lysosomal pH as evident by a decrease in the fluorescence of BioParticles (confocal images, left panel vehicle no effect, right panel cells pre- treated with BafA1 for 30 min, decreased fluorescence). <b>b.</b> Decrease in fluorescence levels of BioParticles following the treatment with BafA1 in monocytes from control and patient CHM4 measured by flow cytometry analysis at 1, 3 and 5 hours following the feeding. <b>c.</b> Representative FACS histograms showing a shift in fluorescence intensity of the CHM and control monocytes fed with BioParticles treated with BafA1 at 1, 3 and 5 h. <b>d.</b> Decreased rate of DQ-ovalbumin degradation in CHM (n = 3) and control (n = 3) patients before and after the treatment with BafA1 measured by flow cytometry analysis at 1, 3 and 5 hours following the feeding. Data expressed as a percent of fluorescence reduction in CHM and control cells treated with BafA1, compared to the non-treated (NT) cells.</p

Experimental design.

<p>Collection of monocyte fractions and culture of primary dermal fibroblasts for the evaluation of gene expression and functional differences between CHM patients and age-matched controls.</p

Mutation in REP-1 affects gene expression and secretion in CHM patients.

<p>a. Hierarchical cluster of 47 probe sets in control and CHM samples. Using consistency testing, twenty-six probe sets were found to be significantly over-expressed and 21 under-expressed in monocytes and primary fibroblasts cells from CHM patients 43 (CHM 6-15) compared to control (Cont 1-5)(p<0.0001, FDR30%) b-d. Level of secretion of the cytokines and growth factors by primary fibroblast cultures into conditioned media. MCP-1, TNF-alpha and FGF factors were detected at significantly higher levels in samples collected from the control cells (n = 9) compared to conditioned media samples from 8 CHM patients (p<0.005)</p

Clinical characteristics of CHM patients and expected effect of determined mutations on the structure of REP-1 protein.

*<p>HM, hand motion; LP, light perception; NLP, no light perception.</p>**<p>Brothers carrying the same mutation in Rep-1 protein.</p>†<p>effect of mutations I553X, L550P, Y504X and P179X previously analyzed by Sergeev et al. 2009.</p

Effect of different mutations on the structure and levels of REP-1 mRNA and protein.

<p><b>a</b>, Effect of different nonsense mutations on the structure of REP-1 protein (Q273X, I460X, M1I and K234X). <b>b</b>, Distribution and position of the mutations in the REP-1 protein, note that 4 of 9 mutations localized in the beta sheet of the REP-1 (blue) and 7 of 9 mutations (P179X, K234X, I244X, I460X, Y504 X, L550P and I553X, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008402#pone-0008402-t001" target="_blank"><b>Table 1</b></a>) localized to domain 2 of the REP-1 protein. <b>c</b>. Levels of mRNA determined by the microarray analysis of the expression profiles from monocytes and fibroblasts from CHM and control patients. Control group, n = 5; group CHM1 includes patients with low levels of REP1 mRNA, n = 7; group CHM2 includes patients with REP-1 mRNA similar to the controls, n = 6. <b>d</b>. Expression levels of REP-1 and REP-2 in different cell types derived from CHM and control patients. Lane: 1, 10 ng of rat recombinant REP-1 or 10 ng of rat recombinant REP-2 with HisTag; cell lysates (40 µg of protein for each) 2, ARPE19; 3, human fetal RPE; 4, MO- monocytes from control; 5, MO-monocytes from patient CHM4; 6, cultured human umbilical vein endothelial cells (HUVECs); 7, primary fibroblasts from control; 8, primary fibroblasts from CHM2 patient. β-actin was used as a loading control.</p