title: Liver histology of an afibrinogenemic patient with the Bbeta-L353R mutation showing no evidence of hepatic endoplasmic reticulum storage disease (ERSD); comparative study in COS-1 cells of the intracellular processing of the Bbeta-L353R fibrinogen vs. the ERSD-associated gamma-G284R mutant.
abstract: BACKGROUND
  Type I fibrinogen deficiencies (hypofibrinogenemia and afibrinogenemia) are rare congenital disorders characterized by low or unmeasurable plasma fibrinogen antigen levels. Their genetic bases are represented by mutations within the three fibrinogen genes. Among the 11 reported missense mutations, a few have been characterized by expression studies and found to have an impaired fibrinogen assembly and/or secretion. Histopathological analyses were previously reported in two hypofibrinogenemic cases with discernible hepatic disease, revealing that both underlying mutations (gamma-Gly284Arg and gamma-Arg375Trp) were associated with hepatic fibrinogen endoplasmic reticulum storage disease (ERSD).
  
  
  OBJECTIVE
  The objective of this study was to investigate the liver histology in an afibrinogenemic patient, homozygous for the Bbeta-Leu353Arg mutation, and to study the intracellular processing of the mutant protein.
  
  
  METHODS
  Liver histology was evaluated by light microscopy, electron microscopy and immunocytochemistry. Intracellular processing of mutant fibrinogen was analyzed by pulse-chase labeling and immunoprecipitation experiments. Messenger RNA levels were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR).
  
  
  RESULTS
  The histopathological characterization of the liver showed no signs of fibrinogen accumulation, a difference from the previously reported findings in two hypofibrinogenemic kindreds with ERSD. To evaluate whether the Bbeta-Leu353Arg mutation and the ERSD-associated gamma-Gly284Arg mutation affected intracellular fibrinogen trafficking differently, both mutant proteins were expressed in COS-1 cells. Bbeta-Leu353Arg led to a more severe secretion defect, but no differences that could explain phenotype-genotype correlation were found in the intracellular processing. Endoglycosidase-H analysis demonstrated a secretion block before translocation to the Golgi medial stacks. Real-time RT-PCR studies showed normal levels of the Bbeta mRNA in the patient's liver.
  
  
  CONCLUSIONS
  The results confirm that Bbeta-Leu353Arg is associated with impaired fibrinogen secretion, but not with hepatic ERSD.
authors.affiliation: Department of Biology and Genetics for Medical Sciences, University of Milan, Milan, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Duga
authors.forename: P
authors.initials: P
authors.lastname: Braidotti
authors.forename: R
authors.initials: R
authors.lastname: Asselta
authors.forename: M
authors.initials: M
authors.lastname: Maggioni
authors.forename: E
authors.initials: E
authors.lastname: Santagostino
authors.forename: C
authors.initials: C
authors.lastname: Pellegrini
authors.forename: G
authors.initials: G
authors.lastname: Coggi
authors.forename: M
authors.initials: M
authors.lastname: Malcovati
authors.forename: M L
authors.initials: ML
authors.lastname: Tenchini
mesh_terms: D000293:Adolescent; D000818:Animals; D001120:Arginine / Q000737:chemistry; D019556:COS Cells; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000473:pathology*; D005340:Fibrinogen / Q000737:chemistry / Q000235:genetics*; D005838:Genotype; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D007930:Leucine / Q000737:chemistry; D008099:Liver / Q000378:metabolism / Q000473:pathology*; D008107:Liver Diseases / Q000235:genetics / Q000473:pathology*; D008297:Male; D008661:Metabolism, Inborn Errors / Q000235:genetics / Q000473:pathology*; D008854:Microscopy, Electron; D009154:Mutation; D020125:Mutation, Missense; D010641:Phenotype; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: C115499:BBeta fibrinogen; D018076:DNA, Complementary; D012333:RNA, Messenger; D012313:RNA; D005340:Fibrinogen; D001120:Arginine; D006026:Glycoside Hydrolases; D007930:Leucine