Activation-resistant homozygous protein C R229W mutation causing familial perinatal intracranial hemorrhage and delayed onset of thrombosis

Abstract

Introduction We describe a family with two first-degree cousins who presented with similar phenotypes characterized by neonatal intracranial hemorrhage and subsequent onset of thrombosis. Patients/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.

Diffuse Intracerebral Hemorrhage in an Infant With a Novel Homozygous Variant Leading to Severe Protein C Deficiency

“…While neonatal purpura fulminans is the hallmark of congenital protein C deficiency, neonatal intracranial hemorrhage is another clinical manifestation that may be a presenting feature of this disorder. One publication describes 2 related patients with a homozygous variant in the protein C gene presenting similar to our patient 15. Severe protein C deficiency could lead to intracranial hemorrhage due to development of microthrombi in vessel capillaries with resulting perivascular hemorrhage.…”

“…One publication describes 2 related patients with a homozygous variant in the protein C gene presenting similar to our patient. 15 Severe protein C deficiency could lead to intracranial hemorrhage due to development of microthrombi in vessel capillaries with resulting perivascular hemorrhage. It has also been proposed that protein C may have a role in maintaining the vascular endothelium in the central nervous system.…”

“…It has also been proposed that protein C may have a role in maintaining the vascular endothelium in the central nervous system. 15 The cranial imaging findings were that of symmetric, diffuse hemorrhage in the subacute phase involving the cerebral white matter. There were no features of active or previous thrombosis.…”

Rational Design of Protein C Activators

“…The mechanism of action of Protac remains elusive and contributes to false negative or positive results and misdiagnosis. For example, Protac based commercial assays fail to diagnose novel PC mutations like T315A and R229W 58 59 . Other potential advantages of FPs over current technologies are noteworthy.…”