title: A missense mutation in the nephrin gene impairs membrane targeting.
abstract: BACKGROUND
  NPHS1, which encodes nephrin, recently has been identified as the gene in which mutations cause congenital nephrotic syndrome of the Finnish type (CNF). We previously reported novel missense mutations of NPHS1 in a Japanese patient with CNF. However, the mechanism by which these missense mutations cause the disorder remains to be clarified.
  
  
  METHODS
  Wild-type nephrin and mutated nephrin complementary DNA were each tagged by the green fluorescence protein (GFP) gene; the expressing vectors of the fusion protein were each transfected to human embryonic kidney 293 cells. We compared intracellular localization of mutated nephrin with that of wild-type nephrin by using GFP and immunostaining examination.
  
  
  RESULTS
  In both wild-type and mutated nephrin (Glu(447)Lys), GFP and immunostaining resulted in a colocalized microgranular pattern along the cell membrane that indicated these recombinant proteins were located at the cell surface. Conversely, in mutated nephrin (Asp(819)Val), GFP aggregation was observed in the cytoplasm, and no fluorescence was observed at the cell membrane, indicating that recombinant mutated nephrin (Asp(819)Val) could not be distributed at the cell membrane and instead was retained in cytoplasm.
  
  
  CONCLUSIONS
  We confirmed that the missense mutation GAC-to-GTC transversion leading to an Asp(819)Val caused the disorder. The present study analyzes in vitro distribution of nephrin with a missense point mutation. The analysis uses a new convenient method, construction of a nephrin-GFP fusion protein.
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics; D003593:Cytoplasm / Q000737:chemistry / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D007668:Kidney; D008164:Luminescent Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D020125:Mutation, Missense; D009404:Nephrotic Syndrome / Q000151:congenital / Q000235:genetics; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals / Q000235:genetics* / Q000502:physiology; D011506:Proteins / Q000032:analysis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
chemical_list: D008164:Luminescent Proteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011506:Proteins; D011993:Recombinant Fusion Proteins; C113267:nephrin; D049452:Green Fluorescent Proteins