title: Activation-resistant homozygous protein C R229W mutation causing familial perinatal intracranial hemorrhage and delayed onset of thrombosis.
abstract: BACKGROUND
  We describe a family with two first-degree cousins who presented with similar phenotypes characterized by neonatal intracranial hemorrhage and subsequent onset of thrombosis.
  
  
  METHODS
  We enrolled the two affected patients, five unaffected family members and fifty-five normal controls. Clinical, laboratory, and radiological characteristics of patients were obtained. Exome sequencing was performed for the older affected child. PROC c.811 C>T was genotyped by PCR in patients, family members, and controls. Protein C amidolytic activity and antigen were measured using the STACHROM® protein C kit and ELISAs. To define functional abnormalities caused by the patients' mutation, recombinant wildtype protein C and its mutants R229W, R229Q and R229A were studied.
  
  
  RESULTS
  For the two cousins, protein C amidolytic activity was 61% and 59% and antigen was 57% and 73% (nl 70-140%), respectively. Exome sequencing revealed a homozygous variant in exon 9 of the protein C (PROC) gene c.811 C>T (R229W). The R229W mutation is located in the calcium binding loop of protein C's protease domain that mediates thrombomodulin interactions. Recombinant R229W-protein C mutant was strikingly defective in rate of activation by thrombin: thrombomodulin, suggesting an in vivo deficit in these children for generation of activated protein C.
  
  
  CONCLUSIONS
  These cases emphasize that protein C and activated protein C are important in maintaining the integrity of the brain vascular endothelium in humans. Moreover, routine protein C assays utilizing snake venom protease fail to detect protein C mutants that are resistant to thrombin:thrombomodulin activation.
authors.affiliation: Department of Pediatrics, College of Medicine, King Saud University, Riyadh, Saudi Arabia. Electronic address: aalsultan1@ksu.edu.sa.
authors.forename: Abdulrahman
authors.initials: A
authors.lastname: Alsultan
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Gale
authors.affiliation: Department of Pediatrics, College of Medicine, King Saud University, Riyadh, Saudi Arabia.
authors.forename: Kadijah
authors.initials: K
authors.lastname: Kurban
authors.affiliation: Department of Pediatrics, College of Medicine, King Saud University, Riyadh, Saudi Arabia.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Khalifah
authors.affiliation: Department of Radiology, College of Medicine, King Saud University, Riyadh, Saudi Arabia.
authors.forename: Fahad B
authors.initials: FB
authors.lastname: Albadr
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA.
authors.forename: John H
authors.initials: JH
authors.lastname: Griffin
mesh_terms: D001921:Brain / Q000098:blood supply / Q000378:metabolism / Q000473:pathology; D006720:Homozygote; D006801:Humans; D007231:Infant, Newborn; D020300:Intracranial Hemorrhages / Q000150:complications / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D010375:Pedigree; D017354:Point Mutation; D011486:Protein C / Q000235:genetics* / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D018180:Thrombomodulin / Q000378:metabolism; D013927:Thrombosis / Q000150:complications / Q000235:genetics* / Q000378:metabolism / Q000473:pathology
chemical_list: D011486:Protein C; D018180:Thrombomodulin; D013917:Thrombin
keywords: Intracranial hemorrhage; Mutation; Neonate; Protein C; Thrombosis