Heterozygous protein C deficiency type I

Protein C is a vitamin K-dependent plasma protein which has anticoagulatory and profibrinolytic properties as a result of inactivating coagulation factors Va and VIIIa and enhancing fibrinolysis. Heterozygous protein C deficiency is well known to be a risk factor for thromboembolic diseases. We here present a family with 16 members deficient in protein C, out of which only two persons were suffering from thromboembolic disorders. In patients suffering from heterozygous protein C deficiency thromboembolic complications in childhood are rare and are not obligatory in adults. These patients should therefore not be treated with oral anticoagulants unless thromboembolic complications have already occurred or are imminent. Coumarin anticoagulation implicates a serious risk of coumarin skin necrosis in protein C deficient patients during the initial therapeutic phase. This risk may be avoided by initiating coumarin therapy with low doses of the drug and in cases of thromboembolic complications by overlapping with heparin anticoagulation.     

Coumarin induced acral skin necrosis associated with hereditary protein C deficiency

Hemorrhagic skin necrosis of the toes was observed in a patient with heterozygous protein C deficiency (protein C:Ag 32% and protein C activity 30%) on the 4th day of coumarin treatment overlapping with effective intravenous anticoagulation with heparin. Family studies revealed protein C deficiency in two sisters of the proposita without a history of thromboembolic disease. Immunologic studies in the proposita at the time of coumarin necrosis revealed slight depression of complement factor C4 and the presence of immune complexes. The present case and review of the literature show that the pathogenetic mechanism leading to coumarin necrosis in patients with protein C deficiency seems not yet to be fully understood.     

Coagulation factor mutations and thrombosis

The coagulation system is governed by a subtle balance between clotting activators and inhibitors. Many genes can contribute to the overall phenotype, and polymorphisms may act to up regulate or down regulate the generation of thrombin, the coagulation-key enzyme. An increase in coagulation factor (gain function) or/and a decrease in coagulation inhibitors (loss of function) may favor venous thromboembolism (VTE). It has been observed since a long time that VTE may be a familial disease, but it was only in 1965 that Egeberg published the first case of inherited antithrombin (AT) deficiency. This was followed by similar reports of protein C (PC) and protein S (PS) deficiencies. Hereditary thrombophilia was thus initially considered as a rare monogenic disorder with incomplete penetrance. AT, PC and PS deficiencies are due to multiple and mostly private mutations of the corresponding genes. Most patients are heterozygous and experience VTE at adult age. Homozygosity associated with severe thrombosis at birth has been observed in newborns with undetectable PC or PS concentrations. The discovery of factor (F) V Leiden and F2 g.20210 G>A, two gain of function mutations, challenged the view of thrombophilia as a rare monogenic disorder. FV Leiden and F2 g.20210 G>A are due to a founder effect and affect populations of European descent with frequencies at 5% and 3% respectively. These two mutations are moderate of risk factor for thrombosis and paved the way for gene-gene and gene-environment interactions. Patients carrying more than one genetic risk factor are at higher risk to develop VTE. The exposition to acquired risk factors such as estrogen based oral contraception may also have a synergistic effect favoring thrombosis in patients with FV Leiden or other genetic risk factors.     

Bilateral transverse sinus thrombosis secondary to a homozygous C677T MTHFR gene mutation

We describe the case of a previously healthy young man who presented with headache, diplopia, nausea, vomiting, and bilateral papilledema. Magnetic resonance venography of the brain revealed thrombosis of the right transverse sinus. Blood tests showed elevated homocysteine levels, and coagulation studies revealed a homozygous C677T mutation and a heterozygous A1298C mutation of the methylenetetrahydrofolate reductase (MTHFR) gene. The patient had no other etiology for venous thrombosis. We recommend screening patients who present with sinus thrombosis for MTHFR gene mutations.     

Genetic analysis of factor V Leiden in a family with history of thrombosis and venous leg ulcers

Inherited resistance to activated protein C has been recognized as a major risk factor for thrombosis. The factor V Leiden mutation, which is detectable by molecular DNA techniques, is responsible for 95% of cases of activated protein C resistance. In our study one patient with venous leg ulcers from a family with a history of thrombosis showed factor V Leiden mutation. Genotypic analysis demonstrated that the patient was homozygous for factor V Leiden. All family members of the index subject showed the same abnormalities. Two were homozygous and 3 were heterozygous for factor V Leiden mutation. The polymerase chain reaction was used to amplify exon 10 of the factor V gene, followed by enzymatic digestion with MnlI for mutation detection. Patients with a family history of thrombosis and factor V Leiden have an increased risk of venous leg ulcers. Screening for factor V Leiden may be indicated in patients with venous leg ulcers and their family members.     

Functional characterization of the protein C A267T mutation: evidence for impaired secretion due to defective intracellular transport

Background  

Activated protein C (PC) is a serine protease that regulates blood coagulation by inactivating coagulation factors Va and VIIIa. PC deficiency is an autosomally inherited disorder associated with a high risk of recurrent venous thrombosis. The aim of the study was to explore the mechanisms responsible for severe PC deficiency in a patient with the protein C A267T mutation by in-vitro expression studies.     

Biosynthesis of a structurally abnormal C2 complement protein by macrophages from C2-deficient guinea pigs

In order to characterize a genetic deficiency of C2 in guinea pigs, production of C2 by peritoneal macrophage cultures derived from four normal, four heterozygous deficient, and four homozygous deficient animals was measured functionally and immunochemically after metabolic labeling with 35S-methionine. Macrophage monolayers from homozygous deficient animals failed to secrete hemolytically detectable C2 up to 74 hr in culture. A single cell hemolytic plaque assay also failed to demonstrate any functional C2 production by cells from homozygous deficient animals. No C2 protein was detected in media from three of the four homozygous deficient animals, but in one, apparent C2 fragments were present. In contrast, intracellular C2 protein was identified in all four homozygous deficient cell cultures. Its mobility on SDS-PAGE was slightly faster than normal. Much less abnormal intracellular C2 protein was recovered from homozygous deficient macrophage monolayers than intracellular C2 protein from normal macrophage monolayers. Monolayers from heterozygous animals produced functional and immunochemical C2 at approximately 30% of the normal rate. Normal rates of biosynthesis and secretion of two other MHC-linked class III antigens, C4 and factor B, were detected in macrophage cultures from homozygous and heterozygous deficient animals. These data suggest that a specific defect, i.e. a structural abnormality in C2 protein, underlies C2 deficiency in guinea pigs.     

Cerebral haemorrhagic infarction in young patients with hereditary protein C deficiency: evidence for "spontaneous" cerebral venous thrombosis.

Among 53 patients with hereditary protein C deficiency belonging to 20 families three women were encountered who, aged 27, 34, and 38 respectively, had had cerebral haemorrhagic infarction, probably due to intracranial venous thrombosis. All three had also had venous thrombosis of the leg and pulmonary embolism either before or after their cerebral infarction. One patient sustained cerebral infarction while receiving an oral contraceptive, but infarction in the two others occurred "spontaneously." One patient also had an intraventricular and subarachnoid haemorrhage during the induction phase of coumarin treatment, which was assumed to have resulted from haemorrhagic infarction of the chorioid plexus, analogous to coumarin provoked haemorrhagic skin necrosis in protein C deficiency. Hereditary protein C deficiency should be considered in young patients with acute or subacute cerebral symptoms, especially if they have a family or personal history of venous thromboembolism.     

Altered gene expression in liver from a murine model of hyperhomocysteinemia

Cystathionine beta-synthase (CBS) deficiency causes severe hyperhomocysteinemia and other signs of homocystinuria syndrome, in particular a premature atherosclerosis with multiple thrombosis. However, the molecular mechanisms by which homocysteine could interfere with normal cell function are poorly understood in a whole organ like the liver, which is central to the catabolism of homocysteine. We used a combination of differential display and cDNA arrays to analyze differential gene expression in association with elevated hepatic homocysteine levels in CBS-deficient mice, a murine model of hyperhomocysteinemia. Expression of several genes was found to be reproducibly abnormal in the livers of heterozygous and homozygous CBS-deficient mice. We report altered expression of genes encoding ribosomal protein S3a and methylthioadenosine phosphorylase, suggesting such cellular growth and proliferation perturbations may occur in homozygous CBS-deficient mice liver. Many up- or down-regulated genes encoded cytochromes P450, evidence of perturbations of the redox potential in heterozygous and homozygous CBS-deficient mice liver. The expression of various genes involved in severe oxidative processes was also abnormal in homozygous CBS-deficient mice liver. Among them, the expression of heme oxygenase 1 gene was increased, concomitant with overexpression of heme oxygenase 1 at the protein level. Commensurate with the difference in hepatic mRNA paraoxonase 1 abundance, the mean hepatic activity of paraoxonase 1, an enzyme that protects low density lipoprotein from oxidation, was 3-fold lower in homozygous CBS-deficient mice. Heterozygous CBS-deficient mice, when fed a hyperhomocysteinemic diet, have also reduced PON1 activity, which demonstrates the effect of hyperhomocysteinemia in the paraoxonase 1 activity.     

A novel homozygous missense mutation in the protein C (PROC) gene causing recurrent venous thrombosis

Summary A novel homozygous CCCCTC (Pro 247 Leu) substitution was detected in the protein C genes of a patient, born to consanguineous parents, with inherited type 1 protein C deficiency and recurrent venous thrombosis. Since one of four heterozygous relatives was also clinically affected, the condition appears to be inherited as an incompletely recessive trait in this family.     

Two different missense mutations at Arg 178 of the protein C (PROC) gene causing recurrent venous thrombosis

Summary Non-identical missense mutations were identified at Arg 178 in the protein C genes of two patients with heterozygous type 1 protein C deficiency and recurrent venous thrombosis.     

Hageman factor deficiency (factor XII)--hemorrhage or thrombosis?

Four generations of a kin with congenital Factor XII deficiency were examined for coagulation and fibrinolysis, with the homozygous female carrier of features with a Factor XII below 1% also revealing certain indications of a disturbed fibrinolysis. The other members of the family had to be evaluated as heterozygous ones, showing values of Factor XII between 40 and 60%. The findings are discussed by referring to data from literature.     

Molecular basis of a selective C1s deficiency associated with early onset multiple autoimmune diseases

We have investigated the molecular basis of selective and complete C1s deficiency in 2-year-old girl with complex autoimmune diseases including lupus-like syndrome, Hashimoto's thyroiditis, and autoimmune hepatitis. This patient's complement profile was characterized by the absence of CH50 activity, C1 functional activity <10%, and undetectable levels of C1s Ag associated with normal levels of C1r and C1q Ags. Exon-specific amplification of genomic DNA by PCR followed by direct sequence analysis revealed a homozygous nonsense mutation in the C1s gene exon XII at codon 534, caused by a nucleotide substitution from C (CGA for arginine) to T (TGA for stop codon). Both parents were heterozygous for this mutation. We used the new restriction site for endonuclease Fok-1 created by the mutation to detect this mutation in the genomic DNA of seven healthy family members. Four additional heterozygotes for the mutation were identified in two generations. Our data characterize for the first time the genetic defect of a selective and complete C1s deficiency in a Caucasian patient.     

A novel nonsense mutation in the protein C (PROC) gene (Trp-29→Term) causing recurrent venous thrombosis

A novel heterozygous TGG→TAG (Trp-29 →Term) substitution was detected in three members of a family with inherited type 1 protein C deficiency and recurrent venous thrombosis.     

Type C Niemann-Pick disease. Abnormal metabolism of low density lipoprotein in homozygous and heterozygous fibroblasts

The esterification of cholesterol derived from human low density lipoprotein (LDL) or fetal bovine serum (FBS) was deficient in cultured fibroblasts from subjects with heterozygous and homozygous type C Niemann-Pick (NPC) disease. Failure to significantly esterify LDL-derived cholesterol resulted in abnormal accumulation of predominantly unesterified cholesterol in homozygous NPC fibroblasts. Compared with normal and homozygous fibroblasts, heterozygous NPC fibroblasts synthesized intermediate levels of cholesteryl ester during the initial 6 h of incubation with LDL. The rate of cholesterol esterification in heterozygous cells was normal when measured over a 24-h period of incubation with LDL. In addition to demonstrating a defect in cholesterol esterification, homozygous NPC fibroblasts accumulated more total cholesterol when incubated with LDL or FBS than normal fibroblasts accumulated. When heterozygous NPC fibroblasts were incubated with LDL or FBS, cellular accumulation of cholesterol reached levels that were high-normal or intermediary between levels observed in normal and homozygous NPC fibroblasts. The partial expression of these metabolic errors in the heterozygous genotype relevantly links these errors to the primary mutation of this disorder.     

Reduced C8 beta messenger RNA expression in families with hereditary C8 beta deficiency

Individuals with functional C8 beta deficiency are at increased risk for systemic neisserial infections. Studies by others have shown that the structural gene for this protein appears intact in deficient individuals. We studied affected individuals from 10 unrelated families to determine the basis for their defect. Using chain-specific antisera, C8 beta was undetectable on immunoblots of their sera. The polymerase chain reaction was used to probe cDNA synthesized from RNA isolated from human liver cells, HepG2 cells, peripheral blood monocytes, and fibroblasts to identify a readily available cell source expressing C8 beta message. Cells from each of these sources expressed C8 beta message. The identity of the amplified product was confirmed and this approach was used to probe cDNA synthesized from RNA harvested from monocytes or fibroblasts obtained from two unrelated families with C8 beta deficiency. C8 beta mRNA was readily detectable in C8 beta sufficient and heterozygous family members but required Southern blotting and hybridization to the 32P-labeled C8 beta probe for detection in the homozygous deficient probands. These results suggest that C8 beta-deficient individuals produce less C8 beta-specific mRNA than do normals and that the underlying basis for this deficiency is an abnormality in intracellular events that precede secretion.     

Complete complement components C4A and C4B deficiencies in human kidney diseases and systemic lupus erythematosus

Although a heterozygous deficiency of either complement component C4A or C4B is common, and each has a frequency of approximately 20% in a Caucasian population, complete deficiencies of both C4A and C4B proteins are extremely rare. In this paper the clinical courses for seven complete C4 deficiency patients are described in detail, and the molecular defects for complete C4 deficiencies are elucidated. Three patients with homozygous HLA A24 Cw7 B38 DR13 had systemic lupus erythematosus, mesangial glomerulonephritis, and severe skin lesions or membranous nephropathy. Immunofixation, genomic restriction fragment length polymorphisms, and pulsed field gel electrophoresis experiments revealed the presence of monomodular RP-C4-CYP21-TNX (RCCX) modules, each containing a solitary, long C4A mutant gene. Sequencing of the mutant C4A genes revealed a 2-bp, GT deletion in exon 13 that leads to protein truncation. The other four patients with homozygous HLA A30 B18 DR7 had SLE, severe kidney disorders including mesangial or membranoproliferative glomerulonephritis, and/or Henoch Schoenlein purpura. Molecular genetic analyses revealed an unusual RCCX structure with two short C4B mutant genes, each followed by an intact gene for steroid 21-hydroxylase. Nine identical, intronic mutations were found in each mutant C4B. In particular, the 8127 g-->a mutation present at the donor site of intron 28 may cause an RNA splice defect. Analyses of 12 complete C4 deficiency patients revealed two hot spots of deleterious mutations: one is located at exon 13, the others within a 2.6-kb genomic region spanning exons 20-29. Screening of these mutations may facilitate epidemiologic studies of C4 in infectious, autoimmune, and kidney diseases.     

A novel single nucleotide polymorphism altering stability and activity of CYP2a6

CYP2A6 is known as a major cytochrome P450 (CYP) responsible for the oxidation of nicotine and coumarin in humans. In this study, we explored genetic polymorphisms, which reduce CYP2A6 activity in Japanese. Two novel mutations in exon 9 of the CYP2A6 gene were found. A single nucleotide polymorphism of T1412C and G1454T resulted in Ile471Thr and Arg485Leu substitution, respectively. The frequency of the former variant allele was considerably high (15.7%), while the latter variant appeared to be a rare polymorphism. Heterologous expression of CYP2A6 using a cDNA possessing C instead of T-base at codon 471 in Escherichia coli caused remarkable reduction of the stability of holoenzyme at 37 degrees C. Furthermore, this variant enzyme almost lacked nicotine C-oxidase activity, although coumarin 7-hydroxylase activity was still observed. These data suggest that individuals homozygous for the T1412C variant allele or heterozygous for this and a defect allele such as the CYP2A6*4 may be poor metabolizer of nicotine, but not coumarin.     

Genetic susceptibility to thrombosis and its relationship to physiological risk factors: the GAIT study. Genetic Analysis of Idiopathic Thrombophilia

Although there are a number of well-characterized genetic defects that lead to increased risk of thrombosis, little information is available on the relative importance of genetic factors in thrombosis risk in the general population. We performed a family-based study of the genetics of thrombosis in the Spanish population to assess the heritability of thrombosis and to identify the joint actions of genes on thrombosis risk and related quantitative hemostasis phenotypes. We examined 398 individuals in 21 extended pedigrees. Twelve pedigrees were ascertained through a proband with idiopathic thrombosis, and the remaining pedigrees were randomly ascertained. The heritability of thrombosis liability and the genetic correlations between thrombosis and each of the quantitative risk factors were estimated by means of a novel variance component method that used a multivariate threshold model. More than 60% of the variation in susceptibility to common thrombosis is attributable to genetic factors. Several quantitative risk factors exhibited significant genetic correlations with thrombosis, indicating that some of the genes that influence quantitative variation in these physiological correlates also influence the risk of thrombosis. Traits that exhibited significant genetic correlations with thrombosis included levels of several coagulation factors (factors VII, VIII, IX, XI, XII, and von Willebrand), tissue plasminogen activator, homocysteine, and the activated protein C ratio. This is the first study that quantifies the genetic component of susceptibility to common thrombosis. The high heritability of thrombosis risk and the significant genetic correlations between thrombosis and related risk factors suggest that the exploitation of correlated quantitative phenotypes will aid the search for susceptibility genes.     

Linkage between C2 deficiency and theHLA-A10,B18,Dw2/BfS haplotype in a French family

A linkage between C2 deficiency and the HLA-A10,B18/BfS antigens has been found in a French family from the Strasbourg area. The propositus, suffering from a chronic glomerulonephritis, is homozygous forHLA-A10,B18/BfS and totally C2-deficient. The parents and the brother are heterozygous for C2 deficiency and share theHLA-A10,B18/BfS haplotype. MLC tests and HLA-D typing revealed that the homozygous C2-deficient patient is also homozygous at theHLA-D locus for the w2 specificity. Evidence was obtained for a heterogeneity of the HLA-Dw2 specificity. This observation confirms the remarkable association between C2 deficiency and theHLA-A10,B18,Dw2 haplotype.