Gene Variants Associated With Venous Thrombosis: A Replication Study in a Brazilian Multicentre Study.

Single nucleotide polymorphisms (SNP) associated with Venous Thromboembolism (VTE) risk have been identified in European and American populations. Replicate SNPs associated with VTE in a Brazilian multicenter case-control study of the Southeast region. Patients with previous VTE assisted at the Outpatient Clinics of 3 centers of the Southeast Brazilian region were compared to normal controls of the same geographic region. We evaluated 29 SNPs associated with VTE risk in other populations, and 90 SNPs for stratification analysis of the population. Due to high admixture of Brazilian population and lack of previous studies, the calculation of the sample power was performed after genotyping. Sample size, allelic frequency and Hardy-Weinberg equilibrium were estimated. The association and odds ratio analyses were estimated by logistic regression and the results were adjusted for multiple tests using Bonferroni correction. The evaluation of the genetic structure similarity in the cases and controls was performed by AMOVA. 436 cases and 430 controls were included. It was demonstrated that this sample has a statistical power to detect a genetic association of 79.4%. AMOVA showed that the genetic variability between groups was 0.0% and 100% within each group. None of the SNPs showed association with VTE in our population. A Brazilian multicenter case-control study with adequate sample power, high genetic variability though no stratification between groups, showed no replication of SNPs associated with VTE. The high admixture of Brazilian population may be responsible for these results, emphasizing the influence of the population genetic structure in association studies.

Folate, vitamin B12 and Homocysteine status in the post-folic acid fortification era in different subgroups of the Brazilian population attended to at a public health care center.

BACKGROUND: Folate and vitamin B12 are essential nutrients, whose deficiencies are considerable public health problems worldwide, affecting all age groups. Low levels of these vitamins have been associated with high concentrations of homocysteine (Hcy) and can lead to health complications. Several genetic polymorphisms affect the metabolism of these vitamins. The aims of this study were to assess folate, vitamin B12 and homocysteine status in distinct Brazilian individuals after the initiation of folic acid fortification by Brazilian authorities and to investigate the effects of RFC1 A80G, GCPII C1561T and MTHFR C677T polymorphisms on folate, vitamin B12 and Hcy levels in these populations. METHODS: A total of 719 individuals including the elderly, children, as well as pregnant and lactating women were recruited from our health care center. Folate, vitamin B12 and Hcy levels were measured by conventional methods. Genotype analyses of RFC1 A80G, GCPII C1561T and MTHFR C677T polymorphisms were performed by PCR-RFLP. RESULTS: The overall prevalence of folate and vitamin B12 deficiencies were 0.3% and 4.9%, respectively. Folate deficiency was observed only in the elderly (0.4%) and pregnant women (0.3%), whereas vitamin B12 deficiency was observed mainly in pregnant women (7.9%) and the elderly (4.2%). Plasma Hcy concentrations were significantly higher in the elderly (33.6%). Pregnant women carrying the MTHFR 677TT genotype showed lower serum folate levels (p = 0.042) and higher Hcy levels (p = 0.003). RFC1 A80G and GCPII C1561T polymorphisms did not affect folate and Hcy levels in the study group. After a multivariate analysis, Hcy levels were predicted by variables such as folate, vitamin B12, gender, age and RFC1 A80G polymorphism, according to the groups studied. CONCLUSION: Our results suggest that folate deficiency is practically nonexistent in the post-folic acid fortification era in the subgroups evaluated. However, screening for vitamin B12 deficiency may be particularly relevant in our population, especially in the elderly.

Increased adhesive properties of neutrophils and inflammatory markers in venous thromboembolism patients with residual vein occlusion and high D-dimer levels.

BACKGROUND: Venous thromboembolism (VTE) develops via a multicellular process on the endothelial surface. Although widely recognized, the relationship between inflammation and thrombosis, this relationship has been mostly explored in clinical studies by measuring circulating levels of inflammatory cytokines. However, the role of inflammatory cells, such as neutrophils, in the pathogenesis of VTE is not clear in humans. AIMS: To evaluate the adhesive properties of neutrophils, erythrocytes and platelets in VTE patients and to correlate findings with inflammatory and hypercoagulability marker levels. METHODS: Study group consisted of twenty-nine VTE patients and controls matched according to age, gender and ethnic background. Adhesive properties of neutrophils, erythrocytes and platelets were determined using a static adhesion assay. Neutrophil adhesion molecules expressions were evaluated by flow cytometry. Inflammatory and hypercoagulability marker levels were evaluated by standard methods. Residual vein occlusion (RVO) was evaluated by Doppler ultrasound. RESULTS: No significant difference could be observed in platelet and erythrocyte adhesion between VTE patients and controls. Interestingly, VTE patients with high levels of D-dimer and RVO, demonstrated a significant increase in neutrophil adhesion, compared to controls and remaining patients. Inflammatory markers (IL-6, IL-8, TNF-α) were also significantly elevated in this subgroup, compared to other VTE patients. Adhesive properties of neutrophils correlated with IL-6 and D-dimer levels. Neutrophils adhesion molecules (CD11a, CD11b and CD18) were not altered in any of the groups. CONCLUSION: These findings not only support the hypothesis of an association between inflammation and hypercoagulability, but more importantly, highlight the role of neutrophils in this process.

Increased ADAMTS13 activity in patients with venous thromboembolism.

Increased levels of inflammatory markers and clotting factors have been related to the pathogenesis and prognosis of venous thromboembolism. In particular, the imbalance between VWF and ADAMTS13 has been described in patients with arterial thrombosis. In this study, 77 patients with previous VTE and 77 matched controls were selected for the evaluation of the inflammatory markers, FVW, ADAMTS 13 and D-dimer. The presences of post-thrombotic syndrome and residual vein obstruction were also assessed in patients. Serum levels of TNF-α and IL-6 were significantly increased in patients compared to controls (median=2.25 vs 1.59 pg/mL, P ≤ 0.001; 1.16 vs 0.98 pg/ml, P=0.013, respectively). Plasma levels and activity of VWF (median=150.25 vs 95.39 U/dL, P ≤ 0.001; 145.26% vs 92.39%, P ≤ 0.001) and ADAMTS 13 (median=1088.84 vs 950.80 ng/mL, P ≤ 0.001; 96.03 vs 83.64%, P ≤ 0.001) were also higher in patients. We further analysed the subgroups of patients with higher risk for VTE recurrence or VTE sequelae, defined as the presence of high D-dimer levels, RVO or PTS. All inflammatory markers were significantly higher in patients with increased D-dimer. The presence of PTS or RVO was not associated with higher inflammatory or coagulation parameters. The increased levels of inflammatory markers and VWF may suggest that there is a persistence of inflammatory activity in patients even at long periods after the VTE episode. In this context, it may be postulated that increased levels of ADAMTS13 could represent a compensatory mechanism against persistently increased levels of VWF. Moreover, increased inflammatory activity was associated with increased D-dimer levels, thus it is possible that this inflammatory activity may also be related to the risk of VTE recurrence.