RESUMO
PURPOSE: This study had the aim of comparing two different methods of analysing dentin sialoprotein (DSP) in the gingival crevicular fluid (GCF): the conventional eLISA approach and a new method involving the use of magnetic micro-beads coated with an antibody specific for DSP prior to eLISA analysis. MATERIALS AND METHODS: GCF was taken from six patients following twelve weeks of orthodontic treatment using paper strips inserted into the mesial and distal sulci of the upper incisors, and analysed using both methods. RESULTS: Statistical analysis of the results using the Mann-Whitney non-parametric test showed that the micro-bead approach conferred more reliability and less variability on the conventional eLISA approach. Furthermore, this method, for the first time, enables the quantification of the DSP in the sample in ng/µl. CONCLUSIONS: The innovative micro-bead/eLISA approach proposed provides a reliable means of quantifying the DSP in the GCF.
RESUMO
BACKGROUND: The homologous coagulation factor X (FX), VII (FVII), IX (FIX) and protein C (PC) display striking differences in the carboxyl-terminus, with that of FX being the most extended. This region is essential for FVII, FIX and PC secretion. OBJECTIVES: To provide experimental evidence for the role of the FX carboxyl-terminus. METHODS: Recombinant FX (rFX) variants were expressed in multiple eukaryotic cell systems. Protein and activity levels were evaluated by ELISA, coagulant and amidolytic assays. RESULTS AND DISCUSSION: Expression of a panel of progressively truncated rFX variants in HEK293 cells revealed that the deletion of up to 21 residues in the carboxyl-terminus did not significantly affect secreted protein levels, as confirmed in HepG2 and BHK21 cells. In contrast, chimeric rFX-FVII variants with swapped terminal residues showed severely reduced levels. The truncated rFX variants revealed normal amidolytic activity, suggesting an intact active site. Intriguingly, these variants, which included that resembling the activated FXß form once cleaved, also displayed remarkable or normal pro-coagulant capacity in PT- and aPTT-based assays. This supports the hypothesis that subjects with nonsense mutations in the FX carboxyl-terminus, so far never identified, would be asymptomatic. CONCLUSIONS: For the first time we demonstrate that the FX carboxyl-terminal region downstream of residue K467 is not essential for secretion and provides a modest contribution to pro-coagulant properties. These findings, which might suggest an involvement of the carboxyl-terminal region in the divergence of the homologous FX, FVII, FIX and PC, help to interpret the mutational pattern of FX deficiency.
Assuntos
Coagulação Sanguínea , Fator X/metabolismo , Hepatócitos/metabolismo , Animais , Cricetinae , Fator X/química , Fator X/genética , Células HEK293 , Células Hep G2 , Humanos , Mutagênese Sítio-Dirigida , Mutação , Tempo de Tromboplastina Parcial , Estrutura Terciária de Proteína , Tempo de Protrombina , Relação Estrutura-Atividade , TransfecçãoRESUMO
Methotrexate (MTX), one of the important pillars in the treatment of different forms of cancer, is associated with the development of hepatotoxicity. The 677C>T variant (rs1801133) in the methylenetetrahydrofolate reductase (MTHFR) gene might affect the development of hepatotoxicity. Results in literature are, however, contradictive. The aim of this study was to evaluate the role of the MTHFR 677C>T polymorphism in MTX-induced hepatotoxicity by analyzing a Dutch cohort of pediatric patients treated with high doses of MTX and subsequently performing a meta-analysis. Ninety-eight patients receiving 542 courses of high-dose MTX were genotyped for the MTHFR 677C>T variant. Hepatotoxicity was evaluated retrospectively according to common terminology criteria for adverse events-National Cancer Institute criteria. The influence of MTHFR 677C>T on hepatotoxicity was examined using a generalized estimating equation (GEE) analysis. A fixed-effect meta-analysis based on this and previous studies investigating the association between the MTHFR 677C>T polymorphism and uniformly coded hepatotoxicity was performed. The GEE analysis showed an increased risk of developing hepatotoxicity for T versus C allele (odds ratio (OR) 1.8; 95% confidence interval (CI) 1.0-3.2, P=0.04). This finding was not supported by the meta-analysis including seven studies and 1044 patients; the OR for the 677T versus C allele was 1.1 (95% CI 0.84-1.5, P=0.25). Heterogeneity between studies was observed, possibly related to differences in MTX dose and leucovorin rescue. In conclusion, in patients with cancer, the MTHFR 677T allele has only a minor role in the development of MTX-induced hepatotoxicity. Observed heterogeneity between studies warrants further study into (tailored) leucovorin rescue.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/genética , Metotrexato/efeitos adversos , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Criança , Feminino , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Masculino , Metotrexato/administração & dosagem , Polimorfismo de Nucleotídeo ÚnicoRESUMO
The first child (proband) of nonconsanguineous Caucasian parents underwent genetic investigation because she was affected with congenital choanal atresia, heart defects and kidney hyposplasia with mild transient renal insufficiency. The direct DNA sequencing after PCR of the CHD7 gene, which is thought to be responsible for approximately 60-70% of the cases of CHARGE syndrome/association, found no mutations. The cytogenetic analysis (standard GTG banding karyotype) revealed the presence of extrachromosomal material on 10q. The chromosome analysis was completed with array CGH (30 kb resolution), MLPA and FISH, which allowed the identification of three 6p regions (6p.25.3p23 × 3): 2 of these regions are normally located on chromosome 6, and the third region is translocated to the long arm of chromosome 10. The same chromosomal rearrangement was subsequently found in the father, who was affected with congenital ptosis and progressive hearing loss, and in the proband's sister, the second child, who presented at birth with choanal atresia and congenital heart defects. The mutated karyotypes, which were directly inherited, are thought to be responsible for a variable phenotype, including craniofacial dysmorphisms, choanal atresia, congenital ptosis, sensorineural hearing loss, heart defects, developmental delay, and renal dysfunction. Nevertheless, to achieve a complete audiological assessment of the father, he underwent further investigation that revealed an increased level of the coagulation factor XIII (300% increased activity), fluctuating levels of fibrin D-dimer degradation products (from 296 to 1,587 ng/ml) and a homoplasmic mitochondrial DNA mutation: T961G in the MTRNR1 (12S rRNA) gene. He was made a candidate for cochlear implantation. Preoperative high-resolution computed tomography and magnetic resonance imaging of the temporal bone revealed the presence of an Arnold-Chiari malformation type I. To the best of our knowledge, this study is the second report on partial 6p trisomy that involves the 10q terminal region. Furthermore, we report the first case of documented Arnold-Chiari malformation type I and increased factor XIII activity associated with 6p trisomy. We present a comprehensive report of the familial cases and an exhaustive literature review.
Assuntos
Anormalidades Múltiplas/genética , Malformação de Arnold-Chiari/genética , Trissomia , Sequência de Bases , Atresia das Cóanas/genética , Cromossomos Humanos Par 6 , Análise Citogenética , Feminino , Cardiopatias Congênitas/genética , Humanos , Cariótipo , Masculino , Fenótipo , Insuficiência Renal/genética , Análise de Sequência de DNA , Translocação GenéticaRESUMO
OBJECTIVE: To investigate the role of pulsed electromagnetic field (PEMF) exposure parameters (exposure length, magnetic field peak amplitude, pulse frequency) in the regulation of proteoglycan (PG) synthesis of bovine articular cartilage explants. METHODS: Bovine articular cartilage explants were exposed to a PEMF (75 Hz; 2 mT) for different time periods: 1, 4, 9, 24 h. Then, cartilage explants were exposed for 24 h to PEMFs of different magnetic field peak amplitudes (0.5, 1, 1.5, 2 mT) and different frequencies (2, 37, 75, 110 Hz). PG synthesis of control and exposed explants was determined by Na2-35SO4 incorporation. RESULTS: PEMF exposure significantly increased PG synthesis ranging from 12% at 4 h to 17% at 24 h of exposure. At all the magnetic field peak amplitude values, a significant PG synthesis increase was measured in PEMF-exposed explants compared to controls, with maximal effect at 1.5 mT. No effect of pulse frequency was observed on PG synthesis stimulation. CONCLUSIONS: The results of this study show the range of exposure length, PEMF amplitude, pulse frequency which can stimulate cartilage PG synthesis, and suggest optimal exposure parameters which may be useful for cartilage repair in in vivo experiments and clinical application.
Assuntos
Cartilagem Articular/metabolismo , Campos Eletromagnéticos , Proteoglicanas/metabolismo , Animais , Cartilagem Articular/crescimento & desenvolvimento , Cartilagem Articular/efeitos da radiação , Bovinos , Técnicas de Cultura/métodos , Proteoglicanas/efeitos da radiaçãoRESUMO
Chronic venous stasis determines red blood cell extravasation and either dermal hemosiderin deposits or iron-laden phagocytes. Several authors have suspected that iron could play a role in the pathogenesis of venous leg ulcers. They hypothesized that local iron overload could generate free radicals or activate a proteolytic hyperactivity on the part of metalloproteinases (MMPs) or else down-regulate tissue inhibitors of MMPs. However, they were unable to explain why iron deposits, visible in the legs of patients with chronic venous disease (CVD), cause lesions in only some individuals, whereas in others they do not. We hypothesized that such individual differences could be genetically determined and investigated the role of the C282Y and H63D mutations of the HFE gene. C282Y mutation significantly increases the risk of ulcer in primary CVD more than six times (OR = 6.69; 1.45-30.8; p = 0.01). Patients carrying the H63D variant have an earlier age of ulcer onset, by almost 10 years (p > 0.004). The increased risk of skin lesion and the early age of onset of the disease in HFE carriers confirm in a clinical setting that intracellular iron deposits of mutated macrophages have less stability than those of the wild type. We hypothesize that the physiologic iron protective mechanisms are affected by the HFE mutations and should be investigated in all diseases characterized by the combination of iron overload and inflammation.
Assuntos
Antígenos de Histocompatibilidade Classe I/genética , Sobrecarga de Ferro/patologia , Proteínas de Membrana/genética , Úlcera Varicosa/genética , Feminino , Proteína da Hemocromatose , Humanos , Sobrecarga de Ferro/fisiopatologia , Masculino , Mutação , Prevalência , Úlcera Varicosa/epidemiologia , Úlcera Varicosa/fisiopatologiaRESUMO
BACKGROUND: Co-inheritance of heterozygous factor V deficiency with FV Leiden enhances the activated protein C resistance (APCR) associated with this mutation, resulting in pseudo-homozygous APCR. The role of FV deficiency in modulating thrombotic risk in this rare condition is poorly understood. METHODS AND RESULTS: We have identified in thrombophilic patients with FV deficiency a novel FV gene mutation (c. 4996G>A), predicting the Glu1608Lys substitution in the A3 domain. The heterozygous mutation was detected in three unrelated patients, two carriers of the FV Leiden mutation, and one of the FVHR2 haplotype. The Glu1608Lys change was also present in two subjects with mild FV deficiency, and absent in 200 controls. The FV1608Lys carriers showed reduced mean FV activity (42% +/- 12%) and antigen (53% +/- 18%) levels and, in Western blot analysis, reduced amounts of intact platelet FV. The restriction fragment length polymorphism (RFLP) study identified two haplotypes underlying the mutation, which suggests that it is recurrent. In heterozygous subjects the amount of FV1608Lys mRNA in white blood cells was similar to that produced by the counterpart alleles (FVWt or FVHR2). Recombinant FV1608Lys (rFV1608Lys), detected by Western blot in the conditioned medium, was indistinguishable from rFVWt and FV antigen and activity were found to be respectively 44% +/- 20% and 13% +/- 4% of rFVWt. CONCLUSIONS: Our data indicate that FVGlu1608Lys predicts a CRM (plasma)/CRMred (cell culture) FV deficiency, and may contribute to thrombophilia in carriers of FV Leiden and FVHR2 haplotype via a pseudo-homozygosity mechanism. Our findings help to define the molecular bases of FV deficiency and thrombophilia.
Assuntos
Deficiência do Fator V/genética , Fator V/genética , Mutação de Sentido Incorreto , Trombofilia/genética , Estudos de Casos e Controles , Análise Mutacional de DNA , Saúde da Família , Feminino , Haplótipos , Heterozigoto , Humanos , Incidência , Leucócitos/química , Masculino , Mutação Puntual , RNA Mensageiro/análise , Receptores de Superfície Celular/genéticaRESUMO
OBJECTIVE: To investigate both the biochemical and the potential morphological changes in bovine cartilage explants following treatment with glucosamine HCl, and to evaluate the capability of glucosamine to counteract the degradation of cartilage induced by catabolic agents such as interleukin-1beta (IL-1beta) and the bacterial lipopolysaccharide (LPS). DESIGN: Bovine articular cartilage explants were treated with increasing doses of glucosamine HCl (0.25-25mg/ml) in the absence or in the presence of IL-1beta or LPS. The release of matrix proteoglycans in the medium, as well as variations in nitric oxide and lactate production were evaluated by standard assays. Proteoglycan synthesis was determined by incorporation of Na(2)-(35)SO(4). Ultrastructural analysis was performed by transmission electron microscopy. RESULTS: Increasing doses of glucosamine (2.5, 6.5, 25mg/ml) induced a dose-dependent decrease in proteoglycan synthesis and in lactate production after 24h treatment. The biochemical changes induced by IL1-beta or LPS appeared to be inhibited by 6.5 and 25mg/ml glucosamine. At these concentrations a decrease in cell viability was observed, which reached over 90% at 25mg/ml. CONCLUSIONS: This study shows that pharmacological doses of glucosamine induce a broad impairment in the metabolic activity of bovine chondrocytes, leading to cell death. The inhibition of the catabolic effects induced by IL1-beta and LPS appears related to glucosamine toxicity. In other experimental models, the same or similar doses of glucosamine have previously been used, without showing any adverse effect. We conclude that, in studying the effects of glucosamine, particular attention should be addressed to the experimental model, the doses and the length of treatment. Published by Elsevier Science Ltd. All rights reserved.
Assuntos
Cartilagem Articular/efeitos dos fármacos , Glucosamina/farmacologia , Proteoglicanas/metabolismo , Animais , Cartilagem Articular/ultraestrutura , Bovinos , Células Cultivadas , Condrócitos/ultraestrutura , Interleucina-1/farmacologia , Lactatos/metabolismo , Lipopolissacarídeos/farmacologia , Microscopia Eletrônica , Óxido Nítrico/biossíntese , Proteoglicanas/biossínteseRESUMO
To investigate simultaneously a defect affecting the protein C/protein S (PC/PS) anticoagulant pathway is possible thanks to a methodological approach (ProC(R) Global; Dade Behring) based on the activation of endogenous plasma PC by a snake venom extract. Factor V (FV) Leiden, the most frequent cause of hereditary thrombosis, is well detected by the test with sensitivity of 100% irrespective of the presence/absence of thrombosis in the subjects investigated. The test is also suited to detect PC or PS defect, but in this case the in vitro impairment of the PC/PS pathway is less pronounced particularly for PS defects (sensitivity for PC and PS defect, 85-100 and 30-90%, respectively). In this study, we hypothesized that the lower sensitivity described for PS defect, compared with those of PC and FV Leiden defects, could also be related to the clinical condition of the subject investigated (symptomatic/asymptomatic) rather than solely to the PS plasma activity/level. Therefore, we analyzed 126 subjects with single congenital defects in the PC/PS pathway: 46 subjects with PS deficiency (26 thrombotic cases and 20 asymptomatic relatives), 40 subjects with PC deficiency (25 thrombotic cases and 15 asymptomatic relatives), and 40 heterozygous FV Leiden subjects (25 thrombotic cases and 15 asymptomatic relatives). By a cut-off of normalized Agkistrodon contortix snake venom ratio of 0.84, the sensitivity in the whole group of cases (sensitivity a) was 76.1, 95.0 and 100%, respectively, for PS, PC and FV Leiden defects. The test failed to detect 11 (23.9%) among the 46 PS-deficient subjects, and all these cases except two belonged to the asymptomatic subgroup (9/20; 45%). Excluding the 20 asymptomatic relatives, the new sensitivity (sensitivity b) for the PS defect was 92.3%. The comparison of the sensitivity in the symptomatic PS cases and in the asymptomatic ones was significantly different (P = 0.010). Among the 40 PC-deficient subjects, only two (5.0%) were not detected by the test and they belonged indifferently to the two subgroups. Finally, none of the 40 FV Leiden heterozygotes were misdiagnosed by the test. These results suggest that in symptomatic PS-deficient cases the test could reflect a post-thrombotic effect and/or reveal potential unidentified prothrombotic influences assessing a prothrombotic risk condition.
Assuntos
Proteína C/análise , Deficiência de Proteína S/sangue , Kit de Reagentes para Diagnóstico/normas , Trombofilia/diagnóstico , Estudos de Casos e Controles , Erros de Diagnóstico , Fator V/análise , Feminino , Humanos , Masculino , Proteína C/genética , Proteína C/metabolismo , Proteína S/análise , Fatores de Risco , Sensibilidade e Especificidade , Trombofilia/sangue , Trombose/sangueRESUMO
Two G-to-A mutations at positions 1691 of the factor V (FV) gene and 20210 of the prothrombin (FII) gene have been associated with an increased risk of venous thromboembolism. We report a thrombosis-prone family in which one subject--the propositus who exhibited combined heterozygous FV G1691A and FII G20210A mutations--showed spontaneous and early clinical onset (at 23 years), recurrences of deep-vein thrombosis and pulmonary embolism. His asymptomatic father carried the FII G20210A substitution and his mother, characterized by an isolated thrombotic episode on occasion of surgery (at 48 years), carried the FV G1691A substitution. In the maternal lineage, one of the propositus' uncles had thrombosis on occasion of a bone fracture (at 65 years) despite the absence of known prothrombotic defects. A sister of the propositus carried the FII G20210A and the brother the FV G1691A mutation. They have been asymptomatic until now. The propositus' two children, 20 and 16 years old, both carry the FV G1691A substitution and have been asymptomatic until now. The plasma levels of FII were higher in carriers of the FII G20210A allele if compared with noncarriers, and the activated protein C resistance phenotype, associated with the FV Leiden mutation, showed a complete correlation with the FV G1691A mutation. Despite the very limited number of thrombotic cases involved in this survey, which does not allow statistically sound conclusions, the data obtained from this family suggest that the synergy of inherited factors and transient risk conditions could play a key role in the occurrence of thrombotic accidents.
Assuntos
Fator V/genética , Mutação Puntual , Protrombina/genética , Trombose Venosa/genética , Adolescente , Adulto , Idade de Início , Idoso , Análise Mutacional de DNA , Saúde da Família , Feminino , Predisposição Genética para Doença/genética , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Recidiva , Trombofilia/sangue , Trombofilia/diagnóstico , Trombofilia/genéticaAssuntos
Hemofilia A/complicações , Doenças de von Willebrand/complicações , Fator VIII/genética , Fator VIII/metabolismo , Saúde da Família , Feminino , Hemofilia A/genética , Humanos , Masculino , Linhagem , Ligação Proteica , Doenças de von Willebrand/genética , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismoRESUMO
The role of a common polymorphism in the factor XIII A-subunit gene (FXIII Val34Leu) has been recently investigated as a protective genetic factor against arterial and venous thrombosis. In addition, the less frequent Leu34 allele has been described as a risk factor for intracerebral hemorrhage. We evaluated the prevalence of this polymorphism by PCR in three case-control studies of patients diagnosed as having primary intracerebral hemorrhage (PCH, n = 130), coronary heart diseases (CHD, n = 240; myocardial infarction/no myocardial infarction, 120/120), and cerebrovascular diseases (CVD, n = 240; cerebral infarction/transient ischaemic attack, 120/120). The matched control groups consisted of patients admitted to the hospital without history of vascular disease. In addition, 200 healthy subjects were investigated. The frequency of the mutated allele (Leu34) was higher in patients with PCH than in controls (33.8% vs. 23.1%, P = 0.009) and lower in CHD and CVD patients compared to controls (18.1% vs. 25.2%, P = 0.010 and 17.3% vs. 24.2%, P = 0.011, respectively). Moreover, among the patients with CHD, the Leu34 allele was underrepresented in cases with myocardial infarction than without (12.9% vs. 23.3%, P = 0.004) and than in controls (12.9% vs. 25.2%, P < 0.001). Similar findings were obtained in patients with CVD comparing the cases with cerebral infarction versus cases with transient ischaemic attack (12.5% vs. 22.1%, P = 0.008) and versus controls (12.5% vs. 24.2%, P < 0.001). Finally, considering altogether the groups of ischaemic patients (CHD and CVD, n = 480), it was noted a trend towards a higher mean age of the clinical onset in homozygotes for the Leu allele than in the wild types (P = 0.078). This study indicates that in our population possession of the FXIII Val34Leu mutation predisposes to the occurrence of primary intracerebral hemorrhage and protects against cerebral and myocardial infarction. A wider modulatory role in the progression and onset of atherothrombotic diseases could be ascribed to FXIII Val34Leu.
Assuntos
Substituição de Aminoácidos , Arteriosclerose/genética , Hemorragia Cerebral/genética , Fator XIII/genética , Genes , Mutação de Sentido Incorreto , Polimorfismo Genético , Trombose/genética , Idade de Início , Alelos , Arteriosclerose/epidemiologia , Estudos de Casos e Controles , Hemorragia Cerebral/epidemiologia , Transtornos Cerebrovasculares/epidemiologia , Transtornos Cerebrovasculares/genética , Comorbidade , Doença das Coronárias/epidemiologia , Doença das Coronárias/genética , Diabetes Mellitus/epidemiologia , Progressão da Doença , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Hiperlipidemias/epidemiologia , Hipertensão/epidemiologia , Ataque Isquêmico Transitório/epidemiologia , Ataque Isquêmico Transitório/genética , Infarto do Miocárdio/epidemiologia , Infarto do Miocárdio/genética , Reação em Cadeia da Polimerase , Subunidades Proteicas , Fatores de Risco , Fumar/epidemiologia , Trombose/epidemiologiaRESUMO
Carriership of the factor V (FV) gene marked by the R2-haplotype, a series of linked polymorphisms encoding several amino acid changes in FV, is associated with mild resistance to activated protein C (APC) and with an increased risk of thrombosis. We compared the functional properties of normal FV(a) and R2-FV(a) in model systems and in plasma. FV and R2-FV were equally well activated by thrombin and expressed identical cofactor activities in prothrombin activation. Rate constants of APC-catalyzed inactivation of FVa and R2-FVa were similar both with and without protein S. However, significant differences were observed between haemostatic parameters determined in plasma from homozygous carriers of the R2-gene (n = 5) and age-matched non-carriers (n = 19). Plasma from R2-carriers contained significantly lower FV levels and the ratio of the two FV isoforms (FV1 and FV2) was shifted in favor of FV1. The FV2/FV1 ratio was 1.4 (95% CI = 1.3-1.5) in homozygous carriers of R2 and 2.8 (95% CI = 2.5-3.1) in controls (p < 0.00001). In an APC resistance test which quantifies the cofactor activity of FV in APC-catalyzed FVIII(a) inactivation, homozygous R2-carriers had significantly lower (p < 0.00001) APC sensitivity ratios (APCsr = 1.54, 95% CI = 1.48-1.60) than controls (APCsr = 2.17, 95% CI = 2.05-2.28). This indicates that R2-FV has reduced cofactor activity in APC-catalyzed FVIII(a) inactivation. The changes of the relative amounts of FV1 and FV2 in carriers of the R2-gene will result in increased thrombin formation in the presence of APC and may provide a mechanistic explanation for the increased thrombotic risk associated with the R2-haplotype.
Assuntos
Fator V/genética , Fator Va/genética , Haplótipos , Resistência à Proteína C Ativada/sangue , Adulto , Testes de Coagulação Sanguínea , Relação Dose-Resposta a Droga , Fator V/metabolismo , Fator V/fisiologia , Fator Va/metabolismo , Fator Va/fisiologia , Fator Xa/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Genótipo , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Proteína S/farmacologia , Protrombina/farmacologia , Trombose/etiologia , Trombose/genéticaRESUMO
BACKGROUND AND OBJECTIVE: Hyperhomocysteinemia, due to a combination of genetic and environmental factors, is considered to be a risk factor for vascular disease. Individuals with the thermolabile variant of methylenetetrahydrofolate reductase (MTHFR), due to homozygous C677T MTHFR gene mutation, have significantly raised plasma levels of homocysteine and may be at increased risk of vascular disease. However, it is still controversial a direct association between C677T homozygosity and the occurrence of vascular disease is still controversial. DESIGN AND METHODS: To clarify the contribution of C677T MTHFR mutation in arterial occlusive disease (AOD) or venous thromboembolism (VTE), we performed a case-controlled study including 160 cases with AOD and 180 cases with VTE attending our referral center and compared them with 200 matched healthy controls. MTHFR gene mutation was evaluated by PCR and odds ratios (OR) and the 95% confidence intervals (CI) were used to estimate the risk for venous or arterial thrombosis. RESULTS: There was a high prevalence of homozygotes for the mutated MTHFR allele among the whole group of cases with arterial disease (OR = 2.35, p = 0.001). Considering the AOD cases with and those without associated risk factors for arterial disease separately the difference remained significant only in the latter group (p = 0.168 and P<0.001 respectively). In contrast, the prevalence of mutated homozygotes among the whole group of cases with VTE was not significantly different from that in the control group (OR = 1.67; p = 0.070). Excluding VTE cases with inherited thrombophilia or with circumstantial risk situations the value increased in both subgroups (OR = 2.26; p = 0.006 and OR = 2.03; p = 0.033 respectively). Considering only VTE cases with neither inherited thrombophilia nor circumstantial risk situations the risk increased further (OR = 2.57; p = 0.017). INTERPRETATION AND CONCLUSIONS: These data suggest that in selected patients homozygosity for the MTHFR mutation increases the risk of both arterial and venous thromboses and that differences in selection criteria for the patient group may be responsible in part for the controversial association of the MTHFR mutation and vascular disease.
Assuntos
Arteriopatias Oclusivas/genética , Hiper-Homocisteinemia/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Mutação Puntual , Trombofilia/genética , Adulto , Idoso , Alelos , Substituição de Aminoácidos , Arteriopatias Oclusivas/epidemiologia , Estudos de Casos e Controles , Feminino , Frequência do Gene , Predisposição Genética para Doença , Testes Genéticos , Genótipo , Humanos , Hiper-Homocisteinemia/epidemiologia , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2) , Pessoa de Meia-Idade , Infarto do Miocárdio/epidemiologia , Infarto do Miocárdio/genética , Razão de Chances , Fatores de Risco , Trombofilia/epidemiologiaRESUMO
Several studies have indicated that mild to moderate hyperhomocystinemia is a common cause of arterial occlusive disease. Whether hyperhomocystinemia per se is an independent risk factor for vein thromboembolism (VTE) is still somewhat controversial. Both genetic and nutritional factors influence plasma homocysteine levels. Therefore, we evaluated plasma total homocysteine (tHcy), folate, and vitamin B12 levels and established, by polymerase chain reaction, the presence of the C677T mutation (A223V) in the methylenetetrahydrofolate reductase (MTHFR) gene in 220 cases with VTE without well-established prothrombotic defects. As a control group, 220 healthy subjects from the same geographic area as the cases were investigated. Hyperhomocystinemia was defined as a plasma tHcy level above the 95th percentile in the controls (18.05 micromol/L). Hyperhomocystinemia was found in 16% of cases (odds ratio=3.59; P<0.001); deficiencies of folate (<2.47 ng/mL) or vitamin B12 (<165 pg/mL), defined as values below the 5th percentile in controls, were found in 17.7% (P<0.001) and 12.3% (P=0.015) of cases, respectively. The homozygous condition for the MTHFR mutation (VV) was present in 28.2% of cases and 17.7% of controls (odds ratio=1.82; P=0.013). Comparing only the idiopathic forms of VTE (n=80/220; 36.3%) with normal controls, individuals with hyperhomocystinemia, or individuals homozygous for MTHFR mutation increased the odds ratios to 4.03 (P=0.005) and 2.11 (P=0.018), respectively. No statistically significant difference was observed in the MTHFR genotype distribution of cases and controls with hyperhomocystinemia (P=0.386); however, the normal MTHFR genotype (AA) appeared in control subjects only when tHcy levels were below the 80th percentile (10.57 micromol/L) of the distribution, whereas in case patients, it was present at the highest tHcy levels. A strong association between mutated homozygosity (VV), low folate levels, and hyperhomocystinemia was found in both groups. We conclude that in patients with VTE who do not have coexisting prothrombotic defects, hyperhomocystinemia increases the risk of developing idiopathic and venous thrombosis; the homozygous condition for the MTHFR mutation confers a moderate risk but, together with low folate levels, it is the main determinant of mild hyperhomocystinemia in normal and thromboembolic populations.
Assuntos
Ácido Fólico/sangue , Homocistina/sangue , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Tromboembolia/sangue , Trombose Venosa/sangue , Adulto , Idoso , Feminino , Genótipo , Humanos , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2) , Pessoa de Meia-Idade , MutaçãoRESUMO
We detected two transversions in two unrelated Italian patients with type 2A von Willebrand disease (VWD): a C to A at nucleotide 8821 and a T to A at nucleotide 8830, resulting in the missense mutations Pro864His and Val867Glu respectively. Both mutations were in the heterozygous form and abolished the BstXI restriction site in exon 28 of the VWF gene. In both mutations plasma VWF multimer pattern improved by antiproteases. Moreover, DDAVP normalized plasma VWF multimers in the Pro864His patient, especially when protease inhibitors were present. These new mutations appear to be of the 2A VWD subtype due to the increased susceptibility to proteases.
Assuntos
Mutação de Sentido Incorreto , Doenças de von Willebrand/genética , Adulto , Éxons/genética , Feminino , Humanos , Masculino , Linhagem , Fator de von Willebrand/genéticaRESUMO
Identifying a defect affecting the protein C/protein S (PC/PS) anticoagulant system, using a single global test, has recently become possible thanks to a new methodological approach based on the activation of endogenous plasma PC by Protac, derived from Agkistrodon Contortix snake venom (ACV). The introduction of a commercial test (ProC Global), ACV-based, provides a useful tool for the screening of thrombotic patients since the most frequent causes of inherited thrombophilia are found in the PC/PS system. The test provides information only on the global activity of the anticoagulant pathway but not on PC and PS activity or on the factor V related conditions (e.g., FV Leiden). The present study shows that by carrying out the test alternating the presence of PC-, PS-, or FV-deficient plasma and using appropriate amounts of ACV, it is possible to increase the specificity of the test to correctly evaluate respectively the PC or PS activities or the activated protein C resistance condition (APC-R). These simple modifications applied to the original commercial test allow to detect exactly, using a single, basic methodology, the principal defects affecting the PC/PS anticoagulant pathway. Furthermore, carrying out the tests on an automated coagulometer, in combination or not with the classic ProC Global assay, it is possible to use a unique reagent profile to simultaneously investigate in the same or different samples, the PC, PS, and APC-R defect.
Assuntos
Resistência à Proteína C Ativada/metabolismo , Testes de Coagulação Sanguínea/métodos , Proteína C/metabolismo , Proteína S/metabolismo , Adulto , Venenos de Crotalídeos , Fator V/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tempo de Tromboplastina Parcial , FenótipoRESUMO
We report a thrombotic family with combined type I antithrombin deficiency and factor V Leiden (factor V-R506Q) in which the proposita, affected by recurrent venous and arterial thrombosis, was also characterized by mild hyperhomocysteinemia (28 micromol/l; normal <18.5 micromol/l). Her two thrombotic sisters, with normal antithrombin levels and factor V molecules, showed hyperhomocysteinemia (51 and 30 micromol/l, respectively). Four other members of the family had the combined antithrombin/factor V Leiden defect and two of them had thrombosis. The common A223V mutation in the methylenetetrahydrofolate reductase gene, responsible for the thermolabile variant of the enzyme, was found to be heterozygous in the proposita; the two sisters were homozygous and heterozygous, respectively. The heterozygous sister also had a high titre of antiphospholipid antibodies (85 units of immunoglobulin G antiphospholipid antibody/ml). Furthermore, low plasma folate levels were found in the three hyperhomocysteinemic subjects of the family. This family with several prothrombotic defects is a clear example of the polyfactorial nature of thrombophilia.
Assuntos
Deficiência de Antitrombina III , Fator V/genética , Homocisteína/sangue , Trombose/fisiopatologia , Adulto , Idoso , Antitrombina III/genética , Feminino , Humanos , Masculino , Mutação , Linhagem , Fenótipo , Fatores de Risco , Trombose/sangue , Trombose/genéticaRESUMO
Hemostatic parameters of 495 beta-thalassemic patients (421 with thalassemia major and 74 with thalassemia intermedia) were analyzed, to assess their association with the described thrombophilic condition and to verify the role of additional risk factors (e.g. persistent postsplenectomy thrombocytosis, insulin dependent diabetes mellitus, estrogen-progestin treatment and atrial fibrillation). The prevalence of thromboembolic accidents was 5.2% and in four patients (15.3%) inherited or acquired predisposing defects were recognized. The incidence of thromboembolic events and the associated relative risk due to hemocoagulative abnormalities in these patients are discussed.