Protein C and protein S assessment in hospital laboratories: which strategy and what role for DNA sequencing?

This paper presents a critical assessment of protein C (PC) and protein S (PS) functional and immunological approaches with regard to DNA sequencing in a large hospital recruitment for thrombosis exploration in more than 1700 consecutive patients. After examination of clinical status and PC and PS phenotype, a genotypic study was implemented for 17 PC-deficient and 28 PS-deficient patients (activity < 70%). Sixty-five percent of the genotyped PC-deficient patients were found to have heterozygous mutations. Among the < 70% values, decreases in PC activity without gene mutation were always slight (mean value 64 +/- 7%) while patients presenting a PC gene mutation had a mean 50 +/- 17% activity (P < 0.05). Among the eight PC mutations found, only one has previously been described. A novel mutation in the promoter region (-1522), located in the HNF-1 site and associated with the Y226H heterozygous mutation, was found in a 9-month-old girl with 4% PC activity. Determination of PS functional activity was considerably improved by contemporaneous measurement of calibration and samples in a single step. Only 50% of the genotyped PS-deficient patients demonstrated heterozygous alterations of the gene. The benefit of sequencing to identify putative causal mutations was only 39% in PS-deficient women, while it was 90% in men. Among the nine PS mutations found, six have not yet been published. In the present paper, we explain our methodological choices and diagnostic strategy.

Chronic disseminated intravascular coagulation and childhood-onset skin necrosis resulting from homozygosity for a protein C Gla domain mutation, Arg15Trp.

A toddler of Haitian descent presented with an 18-month history of chronic consumption coagulopathy, followed by catastrophic skin necrosis. Protein C deficiency (1% to 3% of control) was noted by functional assay; chromogenic assay and antigen levels were 30% of control. Plasma infusion abrogated the disseminated intravascular coagulation-like state. The authors identified a homozygous mutation, C1432T, resulting in a missense, Arg15Trp, in the gamma-carboxyglutamate domain of the protein. Chronic consumption coagulopathy without purpura fulminans or venous thrombosis is a rare presentation of defective protein C pathway. The result of this mutation is a mixed type I (low antigen) and type II (low function) phenotype.

Clinical management of protein C deficiency.

Protein C (PC) is a vitamin K-dependent plasma protein that is structurally similar to other coagulation factors such as prothrombin and Factor X. PC is converted to its active anticoagulant form by a thrombin-thrombomodulin complex on the surface of capillary endothelial cells. Activated PC (APC) prevents the formation of blood clots by specifically inactivating factors Va and VIIIa in the clotting cascade. Both acquired and hereditary forms of PC deficiency exist, with hereditary further categorised as heterozygous, homozygous as well as doubly heterozygous. Patients suffering from symptomatic heterozygous PC deficiency present with purpura fulminans, venous thrombosis and/or pulmonary embolism. Homozygous PC deficiency is usually associated with the development of severe and often fatal, purpura fulminans and disseminated intravascular coagulation (DIC) during the neonatal period. Various therapeutic options have been described for long-term management of severe heterozygous and homozygous PC deficiencies. For the treatment of heterozygous PC deficiency, oral anticoagulation with a coumarin derivative or heparin therapy remains standard therapy. Homozygous patients may be treated with fresh frozen plasma (FFP) and iv. PC concentrate or coumarin derivatives. Other therapeutic options for the treatment of hereditary PC deficiency include the use of low-molecular weight heparin (LMWH), steroids and liver transplantation. Maintenance of a symptom-free life depends on response to therapy. Patients responding well to treatment can expect normalisation of haemostasis as well as improvement of microcirculation and resolution of purpura fulminans.

Single founder mutation (W380G) in type II protein C deficiency in Finland.

The present study investigated the genetic basis for type II protein C deficiency in Finland, where this form has an unusually high incidence. We demonstrated that, first, a single novel mutation W380G in the protein C gene (PROC) explained 25/26 index patients, estimated to represent two thirds of all families with type II deficiency in Finland. Second, extended chromosomal conservation, i.e. a specific haplotype, around the W380G mutation was indicated in unrelated patients. Third, a local geographical origin for the W380G mutation was suggested by genealogical data. These results are in contrast to the heterogeneity in type II protein C deficiency elsewhere, but closely parallel disorders of the Finnish disease heritage. The high frequency of the type II disease can be explained by founder effect and subsequent enrichment of a single mutation in Finland. The present study also provided a simple means for genetic diagnosis of this disease and the genetic test can be included in the routine screenings in this population.

Protein C and S deficiency.

Protein C and S deficiency states predispose affected individuals to thrombosis, especially venous thrombosis of the lower extremities, usually beginning in the teenage years. Treatment of these patients is generally with oral anticoagulation, following initial heparinization. The classification of the deficiency states is dependent upon determination of the quantity and functional activity of either protein.