RESUMEN
BACKGROUND: Somatic mutations in the calreticulin gene (CALR) were recently described in essential thrombocythemia (ET) and primary myelofibrosis with non-mutated JAK2 or MPL. The aim of this single-center study was to compare the clinical and biological features of ET patients according to their mutational status. METHODS: We included 40 patients with ET followed in hematology consultation. The JAK2 V617F mutation was assessed by quantitative PCR. For the detection of CALR mutations, we performed a PCR amplification of CALR exon 9 followed by direct sequencing. RESULTS: Among 40 study patients, 23 (57.5%) harbored V617F JAK2, 12 of the 17 patients without JAK2 mutation harbored CALR, no patient expressed MPL mutation and 5 were negative for all three mutations. Five types of mutations were identified with predominance of 52bp deletion and 5bp insertion (7/12 and 2/12 respectively). The incidence of thrombotic events at diagnosis was significantly higher in JAK2 mutated patients (P<0.05). Biologically, patients with CALR mutation had significantly higher platelet count (P<0.01) and significantly lower hemoglobin level (P<0.05) than those with V617F JAK2 mutation. CONCLUSION: JAK2 and CALR mutation screening in ET has a diagnostic value. Each mutation displays a distinct phenotype with uncertain impact on long-term outcome.
Asunto(s)
Calreticulina/genética , Heterogeneidad Genética , Janus Quinasa 2/genética , Trombocitemia Esencial/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Exones/genética , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutación , Mutación Missense , Fenotipo , Mutación Puntual , Alineación de Secuencia , Análisis de Secuencia de ADN , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Adulto JovenRESUMEN
Hereditary protein S (PS) deficiency is an autosomal disorder caused by mutations in the PS gene (PROS1). Conventional PCR-based mutation detection identifies PROS1 point mutations in approximately 50% of the cases. To verify if gross copy number variations (CNVs) are often present in point mutation-negative hereditary PS deficiency we used multiplex ligation-dependent probe amplification (MLPA) as a detection tool in samples from individuals with a high probability of having true PS deficiency. To this end, DNA samples from nine PS deficient probands with family members (seven type I and two type III) and nine isolated probands (three type I and six type III), in whom PROS1 mutations were not found by DNA sequencing, were evaluated. An independent quantitative PCR (qPCR) was performed to confirm the findings of the MLPA assay. Family members were also tested when DNA was available. Gross abnormalities of PROS1 were found in six out of eighteen probands. In three probands complete deletion of the gene was detected. Two probands had a partial deletion involving different parts of the gene (one from exon 4 through 9 and another from exon 9 through 11). One family showed a duplication of part of PROS1. qPCR analysis was in accordance with these results. In conclusion, this study substantiates that gross gene abnormalities in PROS1 are relatively common in hereditary PS deficient patients and that MLPA is a useful tool for direct screening of CNVs in PROS1 point mutation-negative individuals.
Asunto(s)
Proteínas Sanguíneas/genética , Eliminación de Gen , Duplicación de Gen , Mutación Puntual , Deficiencia de Proteína S/genética , Análisis Mutacional de ADN/métodos , Exones , Salud de la Familia , Femenino , Humanos , Masculino , Mutación , Linaje , Reacción en Cadena de la Polimerasa , Proteína S/genéticaRESUMEN
OBJECTIVE: To characterize the first type II protein S (PS) deficiency affecting the epidermal growth factor (EGF)4 domain, a calcium-binding module with a poorly defined functional role. PATIENTS: The proband suffered from recurrent deep vein thrombosis and showed reduced PS anticoagulant activity (31%), and total, free PS antigen and C4bBP levels in the normal range. RESULTS: Reverse transcription-polymerase chain reaction analysis showed the presence of the IVSg-2A/T splicing mutation that, by activating a cryptic splice site, causes the deletion of codons Ile203 and Asp204. Free PS, immunopurified from proband's plasma, showed an altered electrophoretic pattern in native condition or in the presence of Ca2+. The recombinant PS (rPS) mutant showed reduced anticoagulant (<10%) and activated protein C-independent activities (24-38%) when compared with wild-type rPS (rPSwt). Binding of the rPS variant to phospholipid vesicles (Kd 235.7 +/- 30.8 nM, rPSwt; Kd 15.2 +/- 0.9 nM) as well as to Ca2+-dependent conformation-specific monoclonal antibodies for GLA domain was significantly reduced. CONCLUSIONS: These data aid in the characterization of the functional role of the EGF4 domain in the anticoagulant activities of PS and in defining the thrombophilic nature of type II PS deficiency.
Asunto(s)
Deficiencia de Proteína S/genética , Proteína S/química , Eliminación de Secuencia , Adulto , Calcio/farmacología , Proteínas de Unión al Calcio/genética , Proteína de Unión al Complemento C4b/análisis , Factor de Crecimiento Epidérmico/química , Humanos , Proteína S/análisis , Proteína S/genética , Deficiencia de Proteína S/complicaciones , Deficiencia de Proteína S/etiología , Estructura Terciaria de Proteína/genética , Sitios de Empalme de ARN/genética , Recurrencia , Trombosis de la Vena/etiología , Trombosis de la Vena/genéticaRESUMEN
Small amounts of a variant antithrombin (AT) bearing an Arg-129 to Gln mutation were purified from plasma by means of affinity chromatography on insolubilized heparin at very low ionic strength. As a control, two variant antithrombins, one bearing a Pro-41 to Leu mutation and the other an Arg-47 to His mutation, were purified in the same way. The biochemical characterization of the variants and the kinetic study of thrombin and activated factor X (F Xa) inhibition in the presence of heparin and heparin derivatives suggest that Arg-129 plays a specific role in AT conformation and F Xa inhibition enhancement. Indeed, the purified variant adopted the locked conformation described for AT submitted to mild denaturing conditions (Carrell, R.W., Evans, D.Li. and Stein, P.E. (1991) Nature 353, 576-578) and resembling the latent form of plasminogen activator inhibitor (PAI) (Mottonen, J., Strand, A., Symersky, J., Sweet, R.M., Danley, D.E., Geoghegan, K.F., Gerard, R.D. and Goldsmith, E.J. (1992) Nature 355, 270-273). Moreover, the mutant AT was partially reactivated by heparin for thrombin inhibition, but did not respond to the specific pentasaccharide domain of heparin for F Xa inhibition.
Asunto(s)
Antitrombinas/química , Arginina/química , Inhibidores del Factor Xa , Heparina/farmacología , Antitrombinas/genética , Antitrombinas/aislamiento & purificación , Heparina/química , Humanos , Cinética , Mutación , Oligosacáridos/química , Conformación ProteicaRESUMEN
The protein C (PC) pathway, with its cofactor protein S (PS), is an important natural antithrombotic mechanism. Both PC and PS deficiencies have been implicated in thrombophilia. The molecular basis for hereditary PC and PS deficiencies is highly heterogeneous, with a large spectrum of mutations that have various effects on the expression of the relevant allele. A small subset of patients who are homozygous or compound heterozygous for a PC gene mutation have severe thrombotic complications at birth, whereas onset occurs later in the other cases. Patients heterozygous for a PC or PS gene abnormality may develop recurrent thrombosis during adulthood, with a probability of remaining free of thrombosis of about 50% at age 45. A PC or PS gene defect is associated with the factor V Arg 506 to Gln mutation in 10% to 30% of symptomatic patients, suggesting that clinical expression is controlled by several genes in heterozygous patients.
Asunto(s)
Deficiencia de Proteína C , Proteína C/genética , Deficiencia de Proteína S/genética , Mapeo Cromosómico , Genes/genética , Genes/fisiología , Humanos , Mutación Puntual/genética , Mutación Puntual/fisiología , Proteína C/metabolismo , Deficiencia de Proteína S/metabolismo , Trombosis/genética , Trombosis/fisiopatologíaRESUMEN
The mutations observed in patients with antithrombin and protein C deficiencies are mostly substitutions of one nucleotide, or deletions/insertions of fewer than 10 nucleotides in the exons and intron-exon junctions. These genomic abnormalities result in missense changes (involving aminoacids important for protein folding), aberrant polypeptide chains and/or premature termination codons, or abnormal splicing precluding DNA transcription. The number of mutations so far identified is such that it is difficult to use genomic DNA analysis for diagnostic purpose. However, identification of the gene defect can be useful in well-defined situations, such as the risk of homozygosity, and complex or ambiguous plasma phenotypes, which frequently occur in protein C deficiency. Protein S deficiency, the molecular bases of which have been less extensively studied, is due to micromodifications of the coding sequence in only half the cases investigated so far. The mechanisms involved in the remaining cases remain to be identified.
Asunto(s)
Antitrombina III/genética , Mutación , Proteína C/genética , Deficiencia de Proteína S/genética , Proteína S/genética , Alelos , Deficiencia de Antitrombina III , Cromosomas Humanos Par 1 , Cromosomas Humanos Par 2 , Cromosomas Humanos Par 3 , Bases de Datos Factuales , Genes , Humanos , Mutación Puntual , Deficiencia de Proteína C , Eliminación de SecuenciaRESUMEN
The protein C (PC) pathway, with its cofactor protein S (PS), is an important natural antithrombotic mechanism. Patients with phenotypic PS deficiency may develop recurrent thrombosis during adulthood, with a probability of remaining free of thrombosis of about 50% at age 45. The molecular basis for hereditary PS deficiencies is highly heterogeneous, with a large spectrum of mutations that have various effects on the expression of the relevant allele.
Asunto(s)
Deficiencia de Proteína S/fisiopatología , Proteína S/fisiología , Anticoagulantes/metabolismo , Humanos , Fenotipo , Proteína C/fisiología , Deficiencia de Proteína S/genética , Trombosis/genéticaRESUMEN
In a series of 16 propositi with symptomatic protein S deficiency and a protein S gene mutation, we identified a sporadic case of a novel mutation that probably affects gene expression. The mutation, a G to C transversion leading to the substitution of Ala 484 by Pro, was not found in the protein S gene of the patient's parents. Transmission of the paternal and maternal protein S alleles was apparently normal, on the basis of the frequent polymorphism in exon XV. We also checked the transmission of chromosomal material by analysing protein C gene polymorphisms, beta-globin gene frameworks and four variable number of tandem repeats (VNTRs). By combining the results of these analyses, we were able to rule out nonpaternity and to confirm the de novo nature of the mutation.
Asunto(s)
Deficiencia de Proteína S/genética , Proteína S/genética , Alanina/genética , Alelos , Animales , Bovinos , Humanos , Ratones , Mutación Puntual , Polimorfismo Genético , Prolina/genética , Proteína C/genética , RatasRESUMEN
The association of two missense mutations, a Leu 223 to Phe and an Ile 403 to Met, is described in a family presenting with various protein C deficiency phenotypes. In this family, two subjects were compound heterozygotes with protein C levels of about 25%, the other members being heterozygous for only one of the mutations. The Leu 223 to Phe mutation was also found in 9 members of 3 other families and, in all cases but one, resulted in protein C levels below 60% associated with a high incidence of thrombotic complications. The other mutation, an Ile 403 to Met, was identified in those of the family' members who presented with borderline protein C concentrations. In such a family, the genomic DNA analysis represents the only way to differentiate between the genetic status of each family member. The results highlight the importance of the genotype determination and the poor discriminative power of the plasma assays currently used.
Asunto(s)
Mutación Puntual , Deficiencia de Proteína C , Adolescente , Adulto , Secuencia de Bases , Niño , Análisis Mutacional de ADN , Electroforesis en Gel de Poliacrilamida , Femenino , Genes , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Linaje , Fenotipo , Reacción en Cadena de la Polimerasa , Proteína C/genética , Trombosis/genéticaRESUMEN
The aim of this study was to determine if there is a correlation between the activity of a MoAb as an agonist and its ability to bind to the Fc platelet receptor, Fc gamma RIIa. A polymorphism at amino acid 131 [arginine (Arg) or histidine (His)] of Fc gamma RIIa was first shown to be determinant for MoAb-IgG1 binding on monocytes. To clarify the role of this polymorphism in platelet activation by MoAb-IgG1 we (i) established the Fc gamma RIIa polymorphism at the gene level by adapting the denaturating gradient gel electrophoresis method, (ii) analyzed the binding affinity of the MoAbs to Fc gamma RIIa on platelets from homozygous Arg, homozygous His, and heterozygous Arg/His donors, and (iii) characterized the different reactivities of platelets according to the Fc gamma RIIA polymorphism. Among 167 caucasian donors we found 46% heterozygous Arg/His, 36% homozygous His and 18% homozygous Arg. ALB6, and anti CD9, P256 an anti GPIIb-IIIa, and AP3 an anti-GPIIIa were chosen according to their ability (ALB6, P256) or not (AP3) to activate platelets. These 3 MoAbs-IgG1 bind to Fc gamma RIIa with a stronger affinity for the Arg-form of Fc gamma RIIa, a result which was confirmed with the use of diverse MoAbs directed against various antigens. The different abilities of MoAbs to bind to the two Fc gamma RIIa forms were well correlated to the different platelet responses induced by ALB6 and P256. However, low concentrations of ALB6, which allow full activation of platelets from homozygous Arg donors, as did P256, did not induce any activation of platelets from homozygous His donors, whereas P256 is able to induce a low aggregation. The results further define the respective roles of the antigen and the Fc receptor, depending on the MoAb, and the role of the Fc gamma RIIa polymorphism in platelet activation induced by MoAbs. In addition, the results obtained with MoAbs unable to induce platelet activation provided evidence that the binding of a MoAb on Fc gamma RIIa does not predict its ability to activate platelets.
Asunto(s)
Antígenos CD/genética , Activación Plaquetaria , Polimorfismo Genético , Receptores de IgG/genética , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/sangre , Datos de Secuencia MolecularRESUMEN
We analyzed the protein C gene (PROC) with the denaturing gradient gel electrophoresis (DGGE) scanning strategy in a series of 129 patients with suspected protein C (PC) deficiency (93 with low plasma PC levels and 36 with borderline level). At least one sequence variation was found in 104 of the 129 patients. Thirty-nine sequence variations (found in 72 patients) were already reported detrimental mutations. Thirty-three were novel sequence variations, of which 19 (found in 25 patients) were probably detrimental. Five novel mutations (A1T, R9H, S11R, S12R and K193Q) were associated with qualitative plasma PC deficiency, suggesting or confirming the functional importance of amino acids at these positions. This strategy confirmed the diagnosis of inherited PC deficiency in 79/93 (84.9%) patients with low plasma PC levels and 14/36 (38.8%) patients with borderline values. In order to explain abnormal PC levels observed in patients who did not carry detrimental mutations, screening for the -1654C/T and -1641A/G PROC promoter polymorphisms known to influence plasma PC concentrations was performed. The frequency of the CG allele associated with lower PC concentrations was slightly but not significantly lower in 82 heterozygotes for detrimental PROC gene mutations than in 36 patients with no identified detrimental mutations.
Asunto(s)
Mutación , Deficiencia de Proteína C/genética , Proteína C/genética , Adulto , Femenino , Frecuencia de los Genes , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The protein C Gla domain was studied in six families presenting a type II hereditary deficiency characterized by low activity in a coagulation assay and normal activity in an amidolytic assay. Five of these mutations, previously described by our group, affected Arg-5, Arg-1, Arg 229 and Ser 252. We report here the first natural Glu 7 to Asp mutation in a sixth family. We evaluated the binding of the mutated protein C to H11, a monoclonal antibody (mAb) known to recognize the sequence Phe4 to Arg9 of the Gla domain; the presence of calcium ions suppresses the recognition of this epitope by H11. Mutation of Arg229 to Gln and Ser252 to Asn did not modify the inhibition of protein C binding, whereas the Arg-1 to His mutation resulted in a loss of inhibition in the presence of CaCl2. This suggests that the protein C of this patient shows impaired carboxylation. The protein C from patients bearing the mutations Arg-5 to Trp, Arg-1 to Cys and Glu 7 to Asp bound poorly to H11 mAb, even in the absence of calcium ions. The calcium affinity of the Gla domain was studied by pseudo-affinity chromatography, in which protein C was successively eluted from a Mono Q column by CaCl2 10 mM and NaCl 0.6 M. Protein C from the patient bearing the Arg-5 to Asp mutation had a normal elution profile, suggesting that a modification of the propeptide cleavage site impairs the conformation of the Gla domain but not carboxylation.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Calcio/metabolismo , Proteína C/genética , Estructura Terciaria de Proteína , Ácido 1-Carboxiglutámico/genética , Adulto , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Arginina/genética , Ácido Aspártico/genética , Cromatografía por Intercambio Iónico , Cisteína/genética , Ensayo de Inmunoadsorción Enzimática , Histidina/genética , Humanos , Masculino , Datos de Secuencia Molecular , Mutación , Proteína C/química , Proteína C/metabolismoRESUMEN
Using a monoclonal antibody-based assay, we measured the fibrin degradation product release in the supernatant of plasma clots obtained before and after venous occlusion (VO) in 30 patients with definite or suspected vascular thrombosis (19 definite and 2 suspected deep vein thrombosis, 6 recurrent superficial thrombophlebitis, 3 arterial occlusions of lower limbs). tPA and PAI-1 concentrations were determined using ELISA assays; the post-occlusion values were corrected for haemoconcentration. The increase in tPA during VO was correlated with haemoconcentration (r = 0.74), but 3 patients had ineffective VO (less than 2% increase in proteins). The fibrinolytic response to VO was evaluated using the shortening of the time necessary for the release of 200 micrograms of fibrin degradation products per mg of fibrinogen (delta T 200). Two among the 27 patients with effective VO were bad responders with a delta T 200 less than 3 h (whereas all the others had delta T 200 greater than 10 h). These patients had respectively a deficient tPA release (delta tPA = 1 ng/ml) and an elevated PAI-1 level at rest (33 ng/ml). Several other patients were bad responders in terms of tPA release or of shortening of the euglobulin clot lysis time but they had a normal delta T 200. This plasma clot test reflects the ability of free tPA to bind to fibrin (the amount of which depends on the level of tPA and PAI-1), and may be useful in the diagnosis of a hypofibrinolytic state.
Asunto(s)
Productos de Degradación de Fibrina-Fibrinógeno/metabolismo , Fibrinólisis , Trombosis/sangre , Adulto , Anciano , Constricción Patológica , Femenino , Humanos , Masculino , Persona de Mediana Edad , Inactivadores Plasminogénicos/metabolismo , Activador de Tejido Plasminógeno/metabolismoRESUMEN
A monoclonal antibody (mAb 5A5G2) recognized cleaved plasma protein S (PS) but not uncleaved PS. Interestingly, mAb 5A5G2 did not recognize thrombin-cleaved recombinant PS. Microsequencing of cleaved plasma PS showed a Q-S-T-N amino-terminal sequence, inferring cleavage after the Arg 60 residue. The mAb epitope was located within the sequence encompassing residues 61 to 73, i.e. the carboxy-terminal part of the thrombin-sensitive region (TSR). We used this mAb to develop an ELISA assay to quantify in vivo cleaved PS. In plasma from 10 normal subjects, about 10% of PS was cleaved (7.1% to 15.4%), with a more than 2-fold increase in the corresponding sera. We found increased levels of cleaved PS in 8 patients with disseminated intravascular coagulation (DIC) and decreased levels in 22 patients on long-term oral anticoagulant therapy, whereas cleaved PS levels were similar in 8 hemophiliacs and the 10 normal subjects. Cleaved PS levels did not correlate with prothrombin fragment 1+2 levels released after cleavage by FXa in any of the groups, suggesting that circulating FXa is not the main factor involved in the production of cleaved PS in vivo.
Asunto(s)
Proteína S/análisis , Proteína S/metabolismo , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Proteína S/inmunología , Proteínas Recombinantes/inmunologíaRESUMEN
To further elucidate the molecular basis for hereditary thrombophilia, we screened the protein S active gene in 11 families with type I deficiency, using a strategy based on denaturing gradient gel electrophoresis (DGGE) of all the coding sequences. Fragments with an abnormal DGGE pattern were sequenced, and 5 novel mutations were identified in 8 families. The mutations were a 7-nucleotide deletion in exon II, a 4-nucleotide deletion in exon III, a T insertion in exon VII, a C to T transition transforming Leu 259 into Pro and a T to C transition transforming Cys 625 into Arg in 4 families. These mutations were the only sequence variations found in the propositus' gene exons and co-segregated with the plasma phenotype. A total of 28 members of these 8 families were heterozygous for one of the 5 mutations. Twenty-four (58,5%) of the 41 deficient subjects over 18 years of age had clinical thrombophilia, whereas the 13 subjects under 18 were asymptomatic. Of the 28 subjects, 6 (21,5%) were also found to bear the factor V Arg 506 Gln mutation.
Asunto(s)
ADN/genética , Pruebas Genéticas/métodos , Genoma , Deficiencia de Proteína S/genética , Trombosis/genética , Adolescente , Adulto , Anciano , Secuencia de Bases , Niño , Francia , Humanos , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , Linaje , FenotipoRESUMEN
In 10 patients with nephrotic syndrome (NS), the coagulation inhibitors, the fibrinolytic system and several functions of the fibrinogen-fibrin molecule were studied. Among the coagulation inhibitors, only antithrombin III (AT III) was found decreased and correlated with serum-albumin levels. Venous occlusion test provoked a normal tissue plasminogen activator (tPA) release in all patients. The plasminogen activator inhibitor (PAI) had an increased activity in 5 out of the 10 patients. Thrombin and reptilase times were found abnormal in most patients. The thrombin time (TT) prolongation correlated with serum albumin levels and was corrected by adding purified albumin. The fibrinogen was purified from each of the 10 patients' plasma. Only 2 of them showed abnormal polymerization in purified system, suggesting dysfibrinogenaemia. Other functions (thrombin binding, tPA stimulating activity, lysis by purified plasmin) were found normal except in one of the 2 patients with dysfibrinogenaemia whose fibrinogen lysis by plasmin was delayed. It is concluded that an abnormal fibrinogen molecule is not the most frequent explanation for thrombin time prolongation in NS.
Asunto(s)
Fibrinógeno/aislamiento & purificación , Fibrinólisis , Síndrome Nefrótico/sangre , Adulto , Fibrinógeno/análisis , Glicoproteínas/sangre , Humanos , Activadores Plasminogénicos/antagonistas & inhibidores , Inactivadores Plasminogénicos , Tiempo de Trombina , Activador de Tejido Plasminógeno/sangreRESUMEN
The A4070G polymorphism in exon 13 of the factor V (FV) gene, which replaces His by Arg at position 1299 of the B domain, was recently shown to influence circulating FV levels and to contribute to the activated protein C (APC) resistance phenotype. We examined the impact of this polymorphism in a population of unselected patients with venous thromboembolic disease (VTE). The prevalence of the G4070 (R2) allele was determined in 205 patients and 394 healthy subjects of similar age and sex distribution. Thirty-seven patients (18%) were heterozygous for the R2 allele and 1 (0.5%) was homozygous. Forty-four controls (11.2%) were heterozygous for the R2 allele and 1 (0.2%) was homozygous. Thus, the allelic frequency was significantly higher in the patients with VTE than in the healthy controls, with respective values of 9.5% and 5.8%. The odds ratio was 1.8 (95% CI: 1.1-2.8, p = 0.02), pointing to an increased risk of VTE in carriers of the R2 allele. After excluding subjects with putative or confirmed gene defects (mainly the FV R506Q mutation), the R2 allele was still a risk factor for VTE in the remaining patients, with an odds ratio of 2.0 (95% CI: 1.2-3.5, p = 0.01), demonstrating that this polymorphism is itself a risk factor. This study also confirms that the R2 allele influences APC resistance (APCR) in the absence of the FV R506Q mutation.
Asunto(s)
Deficiencia del Factor V/genética , Factor V/genética , Trombofilia/etiología , Trombosis de la Vena/etiología , Resistencia a la Proteína C Activada/etiología , Adulto , Anticonceptivos Hormonales Orales/efectos adversos , Exones/genética , Factor V/análisis , Deficiencia del Factor V/complicaciones , Deficiencia del Factor V/epidemiología , Femenino , Frecuencia de los Genes , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Polimorfismo Genético , Prevalencia , Recurrencia , Riesgo , Trombosis de la Vena/epidemiologíaRESUMEN
To characterize the putative biochemical modifications induced by the Ser 460 to Pro (Heerlen) mutation in protein S (PS), we expressed both wild-type (wt) and mutated recombinant PS in HEK cells. In SDS-polyacrylamide gels, r-PS Heerlen migrated at 71 kDa whereas r-wt PS migrated at 73 kDa, a difference abolished after deglycosylation by N-glycosidase, suggesting that the Ser 460 Pro mutation abolishes N-glycosylation of Asn 458. The affinity of r-wt PS and r-PS Heerlen for C4b-binding protein (C4b-BP) and for phospholipid vesicles was similar. Neither the enhancement of APC-dependent prolongation of the APTT, nor the specific enhancement of FVa and FVIIIa proteolysis by APC in purified systems was affected by the mutation. However, the Ser 460 Pro mutation induced a slight conformational change in the SHBG domain of the PS molecule, as shown by reduced binding affinity for monoclonal antibodies. The type III phenotype associated with the Heerlen mutation might thus result from a slightly modified rate of synthesis or catabolism. The resulting moderate decrease in the circulating PS concentration may modify the equilibrium between free PS and C4b-BP/PS complexes.
Asunto(s)
Mutación Missense , Proteína S/química , Proteína S/genética , Sustitución de Aminoácidos , Anticuerpos Monoclonales/metabolismo , Afinidad de Anticuerpos/efectos de los fármacos , Especificidad de Anticuerpos/efectos de los fármacos , Calcio/farmacología , Línea Celular , Cromatografía de Afinidad , Glicosilación , Humanos , Fosfolípidos/metabolismo , Unión Proteica , Proteína S/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
This study compared a rapid method to detect the nucleotide mutation 1691 G-->A, responsible for the factor V Arg506-->Gln substitution, with a previously established denaturing gradient gel electrophoresis (DGGE) technique in 136 patients with unexplained thrombosis. The new method comprises amplification of the factor V gene exon 10 with a modified oligonucleotide, permitting the introduction of a cleavage site for the restriction endonuclease HindIII in the fragments bearing the mutation. This simple, rapid, inexpensive and nonisotopic method gave the same results as the DGGE method in all subjects tested.