Joyce Maria Annichino-Bizzacchi

Prevalence of the mutation C677 → T in the methylene tetrahydrofolate reductase gene among distinct ethnic groups in Brazil

Vascular disease is a serious public health problem in the industrialized world, and is a frequent cause of death among the adult population of Brazil. Mild hyperhomocysteinemia has been identified as a risk factor for arterial disease, venous thrombosis, and neural tube defects. Individuals homozygous for the thermolabile variant of methylenetetrahydrofolate reductase (MTHFR-T) are found in 5-15% of the general population and have significantly elevated plasma homocysteine levels which represent one of the genetic risk factors for vascular diseases. We have analyzed the prevalence of individuals homozygous for the MTHFR-T in 327 subjects representing the three distinct ethnic groups in Brazil. The prevalence of homozygotes for the mutated allele MTHFR-T was high among persons of Caucasian descent (10%) and considerably lower among Black (1.45%) and Indians persons populations (1.2%). These data suggest that screening for the MTHFR-T allele should help in identifying individuals with a high risk of vascular disease among populations with a heterogeneous background.

Inherited risk factors for thrombophilia among children with Legg-Calvé-Perthes disease

An inherited tendency to hypercoagulability has been suggested as a cause of vascular thrombosis resulting in Legg-Calvé-Perthes disease (LCPD). Here we carried out an investigation of the most common inherited risk factors for hypercoagulability including the mutation in the factor V gene (factor V Leiden), the transition 20.210G-->A in the prothrombin gene, and also the homozygosity for the 677C-->T transition in the methylenetetrahydrofolate reductase gene (MTHFR). The investigation was carried out among 61 Brazilian children with LCPD, who were compared with 296 individuals from the general population. The prevalence of the factor V Leiden mutation was higher in LCPD patients than in the controls (4.9 vs. 0.7%; p = 0.03). However, no patient had the prothrombin gene variant, and no difference was found between patients and controls when homozygosity for MTHFR-T (3.2 vs. 2.6%: p = 0.64) was determined. These data suggest that in our population, the heterozygosity for factor V Leiden was the only inherited risk factor associated with the development of LCPD.

Polymorphisms in the methylenetetrahydrofolate reductase and methionine synthase reductase genes and homocysteine levels in Brazilian children

Hyperhomocysteinemia is a risk factor for thrombosis, and methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) polymorphisms, folate, and B12 levels could contribute to plasma homocysteine (Hcy) variation. Although well established in adults, few studies have been performed in childhood. In this study, we investigated association of polymorphisms C677T and A1298C in the MTHFR gene and A66G in the MTRR gene with Hcy levels in children. These polymorphisms, as well as Hcy, folate, and vitamin B12 levels were investigated in 220 normal children with ages ranging from 1 to 8 years. Plasma Hcy, folate, and vitamin B12 levels were normal in all children. None of the polymorphisms could be considered an independent risk factor for hyperhomocysteinemia during childhood. The median Hcy levels in 37 children (17%) doubly heterozygous for C677T and A1298C mutations in the MTHFR gene were not different from the other genotypes. However, the association of the different genotypes with Hcy, folate, and vitamin B12 levels demonstrated significant P‐values. The folate levels demonstrated a statistically significant decrease (P = 0.0477) from the C677T mutation in the MTHFR gene (TT genotype) when compared to the other groups. Folate was the only independent risk factor for hyperhomocysteinemia. Thus, monitoring the concentrations of folate would be more helpful for evaluating hyperhomocysteinemia and for preventing cardiovascular disease.

Efficacy and safety of dapsone as a second-line treatment in non-splenectomized adults with immune thrombocytopenic purpura

In adults with immune thrombocytopenic purpura (ITP), steroids are usually proposed as first-line therapy, but long-term complete responses are obtained in no more than 20% of patients. For the remaining patients, splenectomy is considered the treatment of choice, with reported “cure” rates from 60–70%. However, the inherent risks of surgery and sepsis after splenectomy without a guarantee of success justify the search for strategies aimed to avoid splenectomy. Here we retrospectively evaluated the results of dapsone treatment in ITP patients that failed first-line therapy with steroids. These patients received dapsone 100 mg/day for a minimum of 30 days before splenectomy was considered. Efficacy was defined as a sustained rise in platelet counts (>50 × 109/l) clearly attributed to dapsone treatment. Among 52 steroid-dependent or refractory patients, dapsone resulted in sustained increases in platelet counts in 44.2% of patients, after a median follow-up of 21.10 months after treatment initiation. The long-term efficacy of dapsone in this setting is further corroborated by the observation that none of the “responding” patients required splenectomy in the follow-up, compared to 69.0% of the “non-responding” patients. Dapsone-related adverse events were mild and promptly reversed by treatment withdrawal. The results of our retrospective analysis suggest that dapsone is a safe and effective second-line agent for steroid-dependent or refractory ITP patients. Because of its well-known safety profile and low cost compared to other potential second-line treatments for ITP, a trial course of dapsone should be viewed as an attractive option before splenectomy in steroid-dependent of refractory adult ITP patients.

Association of severe haemophilia A and factor V Leiden: report of three cases

Summary. The clinical severity of haemophilia is usually related to residual clotting factor activity. Reports of factor V deficiency or protein C have been described in haemophilic patients and both are involved in the activated protein C pathway. Recently, the poor response to activated protein C due to factor V Leiden (FQ506) has been associated with an increased risk of thrombosis and its incidence in the normal population is given as 2–7%. We describe three cases of severe haemophilia A heterozygous for factor V Leiden and its influence on phenotype.

The impact of the search for thrombophilia risk factors among antiphospholipid syndrome patients with thrombosis

Thrombosis is a major clinical feature of the antiphospholipid syndrome. Interactions between genetic and acquired factors could contribute to thrombosis development. In this study, we evaluated 40 patients with antiphospholipid syndrome and thrombosis, 31 primary and nine secondary to systemic lupus erythemathosus, to estimate the carrier rates of factor V Leiden, 20210A ← G prothrombin variant and 677C ← T in the MTHFR gene. Protein C, protein S and antithrombin were measured in 30 patients, with a median of 100.66 ± 23.86, 93.57 ± 36.44 and 98.8 ± 5.67%, respectively. None of the patients were deficient on these natural anticoagulants. No significant variation was found between the patient group and the controls, regarding the prevalence of homozygotes for the mutated 677T allele (2.5 versus 5.4%), or heterozygotes for factor V Leiden (0 versus 0.7%). Despite the fact that these mutations are relatively common in Brazilian thrombophilic patients, its low prevalence in this cohort of patients suggest that these genetic alterations are not risk factors for thrombosis in antiphospholipid syndrome. The prevalence of the mutated allele 20210A of the prothrombin gene was higher in patients when compared with controls (5 versus 0.7%; P = 0.01), suggesting that prothrombin variant could increase the risk of thrombosis in patients with antiphospholipid syndrome.

Assessing the Coagulation Factor Levels, Inherited Thrombophilia, and ABO Blood Group on the Risk for Venous Thrombosis Among Brazilians

Increased coagulation factor levels have been demonstrated to be a risk factor for venous thromboembolism in patients of Caucasian origin. Coagulation factors, hereditary thrombophilia, and ABO blood group were evaluated for venous thrombosis risk in a heterogeneous Brazilian population consisting of 122 women and 53 men, with a median age of 36 years (range 13-63), matched to a control group by age, sex, and ethnicity. Increased levels of factor VIII (odds ratio [OR], 3.1; 95% confidence interval [CI], 1.6-6.0), von Willebrand factor (OR, 2.8; 95% CI, 1.4-5.4), non-O blood group (OR, 2.1; 95% CI, 1.3-3.4), and thrombophilia (OR, 3.4; 95% CI, 1.6-7.1) emerged as independent risk factors for venous thromboembolism. The interaction of high levels of factor IX and factor XI with other independent variables increased the potential for thrombosis synergistically. Therefore, the ability of identifying underlying thrombophilia risk factors in our population was enhanced by the inclusion of these factors in the prothrombotic laboratory workup.

Imbalances in serum angiopoietin concentrations are early predictors of septic shock development in patients with post chemotherapy febrile neutropenia.

BackgroundFebrile neutropenia carries a high risk of sepsis complications, and the identification of biomarkers capable to identify high risk patients is a great challenge. Angiopoietins (Ang -) are cytokines involved in the control microvascular permeability. It is accepted that Ang-1 expression maintains endothelial barrier integrity, and that Ang-2 acts as an antagonizing cytokine with barrier-disrupting functions in inflammatory situations. Ang-2 levels have been recently correlated with sepsis mortality in intensive care units.MethodsWe prospectively evaluated concentrations of Ang-1 and Ang-2 at different time-points during febrile neutropenia, and explored the diagnostic accuracy of these mediators as potential predictors of poor outcome in this clinical setting before the development of sepsis complications.ResultsPatients that evolved with septic shock (n = 10) presented higher levels of Ang-2 measured 48 hours after fever onset, and of the Ang-2/Ang-1 ratio at the time of fever onset compared to patients with non-complicated sepsis (n = 31). These levels correlated with sepsis severity scores.ConclusionsOur data suggest that imbalances in the concentrations of Ang-1 and Ang-2 are independent and early markers of the risk of developing septic shock and of sepsis mortality in febrile neutropenia, and larger studies are warranted to validate their clinical usefulness. Therapeutic strategies that manipulate this Ang-2/Ang-1 imbalance can potentially offer new and promising treatments for sepsis in febrile neutropenia.

Novel insights into the development of atherosclerosis in hemophilia A mouse models

Summary.  Background: Cardiovascular diseases in aging people with hemophilia (PWH) represent a growing concern. The underlying hypocoagulability probably provides a protective effect against acute thrombus formation, but the limited data available show no preventive effect against the development of atherogenesis in PWH. Atherosclerosis‐prone mice are attractive tools for the study of atherosclerosis development, and may provide insights into disease progression in PWH. Methods: Severe hemophilia A (factor VIII‐deficient [FVIIIo]) mice were crossed with mice lacking apolipoprotein E (ApoE−/−) or mice lacking the LDL receptor (LDLR−/−), and then compared to hemostatically normal littermate controls. After mice had received atherogenic diets for 8, 22 or 37 weeks, atherosclerotic lesion size and phenotypic characterization were analyzed in the aortic sinus and whole aortas. Results: ApoE−/−/FVIIIo mice showed a time‐dependent protective effect against the development of atherosclerosis, beginning after 22 diet‐weeks and persisting to 37 diet‐weeks in both the aorta sinus and whole aorta as compared with ApoE−/− mice. Notably, the FVIII deficiency did not influence the progression of atherosclerosis in the FVIIIo/LDLR−/− model as compared with controls at early or late time points. Conclusions: Hypocoagulability ameliorates vascular disease in the ApoE‐deficient model in a lipid‐independent manner. Interestingly, FVIII deficiency did not affect the development of atherosclerosis in LDLR−/− mice. In contrast to the ApoE model, the LDLR model resembles the lipid profile that is commonly observed in humans with atherosclerosis. These findings, to a certain extent, support the notion of atherosclerosis development in the complete absence of FVIII.

Time-course of sFlt-1 and VEGF-A release in neutropenic patients with sepsis and septic shock: a prospective study

BackgroundSeptic shock is the most feared complication of chemotherapy-induced febrile neutropenia. So far, there are no robust biomarkers that can stratify patients to the risk of sepsis complications. The VEGF-A axis is involved in the control of microvascular permeability and has been involved in the pathogenesis of conditions associated with endothelial barrier disruption such as sepsis. sFlt-1 is a soluble variant of the VEGF-A receptor VEGFR-1 that acts as a decoy receptor down-regulating the effects of VEGF-A. In animal models of sepsis, sFlt-1 was capable to block the barrier-breaking negative effects of VEGF-A and to significantly decrease mortality. In non-neutropenic patients, sFlt-1 has been shown to be a promising biomarker for sepsis severity.MethodsWe prospectively evaluated concentrations of sFlt-1 and VEGF-A at different time-points during febrile neutropenia, and evaluated the association of these levels with sepsis severity and septic shock development.ResultsNeutropenic patients that evolved with septic shock (n = 10) presented higher levels of sFlt-1 and VEGF-A measured 48 hours after fever onset than patients with non-complicated sepsis (n = 31) and levels of these biomarkers correlated with sepsis severity scores. Estimation of the diagnostic accuracy of sFlt-1 levels for the discrimination of patients that evolved to septic shock yielded promising results in our study population.DiscussionOur data suggest that sFlt-1 and VEGF-A could be useful biomarkers for sepsis severity in patients with febrile neutropenia. In addition, the kinetics of sFlt-1 release in patients that evolve to septic shock suggest that the sFlt-1 could be a salvage compensatory mechanism in patients with septic shock, but that the magnitude of the sFlt-1 release observed in human sepsis is not sufficient to reproduce the beneficial anti-VEGF-A effects observed in animal models of sepsis.