Activation-Resistant Homozygous Protein C R229W Mutation Causing Familial Perinatal Intracranial Hemorrhage

Two first-degree cousins presented with similar phenotype characterized by neonatal intracranial hemorrhage and subsequent onset of thrombosis at the age of four months. One girl, now 9 yr old, was a full term baby presenting at 13 days of age with seizures and brain CT showing intracranial hemorrhage. At 4 months of age, she presented with multiple thrombotic lesions over both hands and feet that required extensive debridement and improved with anticoagulation and plasma replacement. She has been on enoxaparin with no new lesions afterward. At 9 years of age, protein C amidolytic activity (STACHROM^® protein C) was 61% (nl 70-140%) and protein C antigen (ELISA) was 57% (nl 70-140%). Thrombophilia work up was otherwise normal. The second child is a 1 yr old boy who was delivered by cesarean section because of fetal distress. The mother gave history of decreased fetal movement for few weeks prior to his birth and was noted to be hypotonic. Brain MRI in the first week of life showed massive subacute hemorrhage in the brain parenchyma and upper spinal canal. Bleeding stopped with appropriate supportive care. He did not have purpura fulminans or any evidence of thrombosis during the neonatal period. At 4 months of age, a ventriculoperitoneal shunt was placed because of hydrocephalus and post operatively he developed thrombotic lesion in his left foot that resolved after starting enoxaparin and plasma replacement. He remains on enoxaparin with no new significant lesions. At 10 months of age, protein C amidolytic activity was 59% and protein C antigen was 73%. Both patients suffer from severe permanent neurological deficit and blindness. Dx of protein C homozygous deficiency was established after exome sequencing of the older child revealed a homozygous variant in exon 9 of PROC gene c.811 C>T (R271W equivalent to R229W in mature protein C numbering). Sanger sequencing validated this mutation. Both patients were homozygous (T/T) while tested parents and two siblings were heterozygous (C/T), and 55 controls were normal (C/C). No family history of thrombosis among family members who are heterozygous for this mutation was found. Complete exome sequence analysis also showed no mutations for ATIII, protein S, factors V or VIII, prothrombin, plasminogen, protein Z, or any ADAMTS genes. The R229W mutation is located in the calcium binding loop of protein C's protease domain that mediates thrombomodulin (TM) interactions. To define functional abnormalities caused by the patients' mutation, recombinant protein C mutants R229W, R229Q and R229A were studied. Each was strikingly defective in rate of activation by thrombin:TM, showing an activation rate that was only 1%, 2% and 4%, respectively, compared to wildtype protein C. Once activated, each activated protein C (APC) mutant was moderately affected, showing a 50% reduction in anticoagulant activity and in rate of cleavage of purified factor Va at Arg506. The amidolytic activities were less affected and close to wildtype APC. The plasma half-lives were similar to wildtype APC. These properties of recombinant R229W protein C strongly suggest a severe in vivo deficit in these children for generation of APC, implying a severe deficiency of APC in spite of half-normal levels of protein C zymogen. As routine coag assays utilize snake venom protease, they fail to detect TM-activation-resistant protein C mutants. In summary, homozygous protein C R229W mutation in two related cousins was associated with significant leaky brain blood vessels in the perinatal period and significant neurological insult even prior to the first evidence of clinical thrombosis. This highlights the importance of protein C and APC in maintaining the integrity of the brain vascular endothelium in humans.