Fig 1.
Clinical characteristics of the patient.
(A) Photograph showing skin and hair color of the patient (B) Iris transillumination: 1, normal right iris; patient’s iris with transillumination defects (2, right eye; 3, left eye); Fundus: 4, normal fundus right eye; patient’s poorly pigmented fundi with peripapillary atrophy and foveal hypoplasia (5, right eye; 6, left eye) (C) Electron microscopy (EM) findings of the thin sections of platelets imaged by transmission EM, showing platelet from control (left panel) and patient (right panel) (D) whole mount EM of platelets showing lack of delta-granules in patient (right panel) compared to control (left panel) (E) Computed tomography scan of the chest shows no evidence of pulmonary fibrosis (left panel); right panel showing foamy appearance of alveolar macrophages in bronchoalveolar lavage fluid.
Fig 2.
Molecular and expression analysis.
(A) Chromatogram showing intronic mutation (c.285-10A>G) in genomic level (upper panel) and cDNA level (middle panel) of the patient. Lower panel shows normal cDNA sequence from control for comparison with patient. (B) Relative quantification of mRNA expression (three different isoforms) in patient derived dermal fibroblasts, compared to control. Error bar represents standard deviation from triplicates (*: p<0.05, **: p<0.01). (C) Western blotting results showing the expression of HPS5 in patients compared to control. Two different controls (Control-1 and Control-2), two additional patients with HPS5 (HPS5-1 and HPS5-2), and our proband were included. The level of protein expression was normalized with β-actin.
Fig 3.
Cellular distribution of lysosomes.
A. Distribution pattern CD-63 (green) in three HPS-5 patients including proband. Phalloidin (red) highlights cell boundary and DAPI (blue) stains nucleus. Upper panel shows lower magnification images showing perinuclear accumulation of CD63 in patients. Lower panel represents the higher magnification focusing on the peripheral region. B. Upper panel shows representative images for CD63 staining in 96-well format for quantification. Control image is a representative of three control cell lines used. CD63 is stained green. Lower panel is histogram representing the quantification of mean fluorescence intensity in samples.; ~6000 cells per well were seeded, and three replicates per cell line were measured. Error bars represent standard error of mean; no statistically significant differences were seen.
Fig 4.
LysoTracker red dye staining shows the distribution of acidic organelles including mature lysosomes of two HPS-5 patients, including the proband, compared to control. Cell boundaries are marked with white dotted line. Scale bar represents 200 px (pixels). Representative images for lysotracker staining in 96-well format for quantification. Control image is a representative of three control cell lines used. Lysotracker is stained red. A histogram representing the quantification of mean fluorescence intensity of samples is shown in the loer panel. ~6000 cells per well were seeded, and three replicates per cell line were measured. Data from control samples were pooled. Error bars represent standard error of mean; there were no statistically significant differences.
Table 1.
HPS5 mutations reported in the literature.