An immunoblotting assay to facilitate the molecular diagnosis of Hermansky-Pudlak syndrome.
Hermansky-Pudlak syndrome (HPS) comprises a constellation of human autosomal recessive disorders characterized by albinism and platelet storage pool deficiency. At least eight types of HPS have been defined based on the identity of the mutated gene. These genes encode components of four ubiquitously expressed protein complexes, named Adaptor Protein (AP)-3 and Biogenesis of Lysosome-related Organelles Complex (BLOC)-1 through -3. In patients of Puerto Rican origin, the molecular diagnosis can be based on analysis of two founder mutations. On the other hand, identification of the HPS type in other patients relies on the sequencing of all candidate genes. In this work, we have developed a biochemical assay to minimize the number of candidate genes to be sequenced per patient. The assay consists of immunoblotting analysis of extracts prepared from skin fibroblasts, using antibodies to one subunit per protein complex. The assay allowed us to determine which complex was defective in each of a group of HPS patients with unknown genetic lesions, thus subsequent sequencing was limited to genes encoding the corresponding subunits. Because no mutations within the two genes encoding BLOC-3 subunits could be found in two patients displaying reduced BLOC-3 levels, the possible existence of additional subunits was considered. Through size-exclusion chromatography and sedimentation velocity analysis, the native molecular mass of BLOC-3 was estimated to be 140+/-30 kDa, a value most consistent with the idea that BLOC-3 is a HPS1HPS4 heterodimer (approximately 156 kDa) albeit not inconsistent with the putative existence of a relatively small third subunit.
Nazarian
Ramin
R
Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.
Huizing
Marjan
M
Helip-Wooley
Amanda
A
Starcevic
Marta
M
Gahl
William A
WA
Dell'Angelica
Esteban C
EC
D033963:Adaptor Protein Complex 3 / Q000235:genetics / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D015153:Blotting, Western / Q000379:methods*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D004247:DNA / Q000235:genetics; D022861:Hermanski-Pudlak Syndrome / Q000145:classification / Q000175:diagnosis* / Q000235:genetics*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000276:immunology; D011506:Proteins / Q000235:genetics / Q000276:immunology; D017386:Sequence Homology, Amino Acid
D033963:Adaptor Protein Complex 3; C104378:BLOC1S1 protein, human; C492647:BLOC1S2 protein, human; C509457:BLOC1S3 protein, human; D002352:Carrier Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D004247:DNA