A BLOC-1 mutation screen reveals a novel BLOC1S3 mutation in Hermansky-Pudlak Syndrome type 8

Abstract

Hermansky-Pudlak Syndrome (HPS) is a genetically heterogeneous disorder of lysosome-related organelle biogenesis and is characterized by oculocutaneous albinism and a bleeding diathesis. Over the past decade, we screened 250 patients with HPS-like symptoms for mutations in the genes responsible for HPS subtypes 1-6. We identified 38 individuals with no functional mutations, and therefore, we analyzed all eight genes encoding the biogenesis of lysosome-related organelles complex-1 (BLOC-1) proteins in these individuals. Here, we describe the identification of a novel nonsense mutation in BLOC1S3 (HPS-8) in a 6-yr-old Iranian boy. This mutation caused nonsense-mediated decay of BLOC1S3 mRNA and destabilized the BLOC-1 complex. Our patient's melanocytes showed aberrant localization of TYRP1, with increased plasma membrane trafficking. These findings confirm a common cellular defect for HPS patients with defects in BLOC-1 subunits. We identified only two patients with BLOC-1 defects in our cohort, suggesting that other HPS genes remain to be identified.

Figure 1

Clinical aspects of HPS-8 (bottom panels) compared with controls (top panels). (A) The patient has fair skin and hair compared to other family members. (B) The patient’s hair shaft shows significant pigmentation compared to that a dark haired/ethnically matched control, and no abnormal pigment clumping. (C) The patient has decreased retinal pigment in the periphery compared to control. (D) Whole-mount electron microscopy of the patient’s platelets revealed no delta granules, which are present in control platelets (arrows). (E) A blood smear revealed no abnormal large granules in the neutrophils. (F) Packed melanocytes show reduced pigment in the HPS-8 patient compared to a matched control.

Figure 2

Molecular studies in the HPS-8 patient. (A) Sequencing chromatograms from control and patient genomic DNA. The patient is homozygous for c.131C>A in BLOC1S3 causing p.S44X at the protein level. (B) SNP-array data of chromosome 19 for the HPS-8 patient. The vertical red dotted line shows the position of BLOC1S3 and the red brackets show extended regions of homozygosity (top plot). On the B allele plot (upper chart), the middle dots represent heterozygous SNPs (AB), and the top and bottom borders represent homozygous SNPs (AA or BB). The log R ratio plot (lower chart) shows that there are normal SNP calls in both homozygous regions, indicating that no deletions or insertions are present.

Figure 3

BLOC1S3 mRNA and protein analysis of HPS-8 cells. (A) Quantitative real-time PCR results for BLOC1S3 mRNA expression in patient compared to control fibroblasts and melanocytes. Values shown are percentage expression of BLOC1S3 in patient cells compared to control cells, normalized by GAPDH (Error bars = ± 1 SEM, n=3, p<0.001). (B) Schematic diagram depicting the exon structure of BLOC1S3, where only exon 2 is protein coding, and contains both the start (ATG; M) and stop (X) codons (grey box). Arrowheads show the position of the PCR primer used for (C) and arrow shows position of the HPS-8 patient’s mutation. (C) Standard PCR on cDNA of control and patient’s fibroblasts or melanocytes. Agarose gel images of PCR products show no detectable BLOC1S3 amplification in patient’s cDNA for either primer set (coding: F1-R; 5′UTR: F2-R); GAPDH was amplified. NTC, non-template control. (D) Immunoblots of fibroblast extracts from the HPS-8 patient and the previously reported HPS-9 patient for the BLOC-1 subunits cappuccino, dysbindin, pallidin and snapin. Cappuccino and dysbindin (and pallidin in the HPS-9 patient) are undetectable. Pallidin is reduced in the HPS-8 patient and snapin is also reduced in both BLOC-1 patients. (E) Immunoblots of lysates from HPS-8 and HPS-9 (BLOC-1), HPS-4 (BLOC-3) and HPS-5 (BLOC-2) patients using antibodies to the HPS4 and HPS5 proteins, showing independent assembly of the BLOC complexes. Loading was controlled by immunoblotting the same membrane for β-actin.

Figure 4

Common defect of TYRP1 trafficking in BLOC-1 patients. (A) Confocal immunofluorescence images showing TYRP1 in the dendrites and tips of control melanocytes while in the HPS-8 cells the TYRP1 appears to be more perinuclear, in the plasma membrane, and co-localizes with the Golgi (inserts). (B) Tyrosinase localization appears normal in the HPS-8 patients’ melanocytes and is comparable with control cells. Golgi marked by TGN46, nuclei stained with DAPI and scale bar represents 20μm. (C) Plasma membrane biotinylation assay shows increased TYRP1 protein on the membrane of the HPS-8 patients cells compared to control cells despite there being less TYRP1 in the whole cell lysate from the patient. Blotting the same membrane for β-actin demonstrates purity of the membrane fraction, and equal loading of the whole cell lysate. (D) TYRP1 internalization assay shows a decreased rate of endocytosis from the plasma membrane in the HPS-8 patient cells compared to that of control. Values shown are percentage of starting TYRP1 on the plasma membrane (error bars = ± 1 SEM, n=3).