Chromatin regulation by Histone H4 acetylation at Lysine 16 during cell death and differentiation in the myeloid compartment.

Histone H4 acetylation at Lysine 16 (H4K16ac) is a key epigenetic mark involved in gene regulation, DNA repair and chromatin remodeling, and though it is known to be essential for embryonic development, its role during adult life is still poorly understood. Here we show that this lysine is massively hyperacetylated in peripheral neutrophils. Genome-wide mapping of H4K16ac in terminally differentiated blood cells, along with functional experiments, supported a role for this histone post-translational modification in the regulation of cell differentiation and apoptosis in the hematopoietic system. Furthermore, in neutrophils, H4K16ac was enriched at specific DNA repeats. These DNA regions presented an accessible chromatin conformation and were associated with the cleavage sites that generate the 50 kb DNA fragments during the first stages of programmed cell death. Our results thus suggest that H4K16ac plays a dual role in myeloid cells as it not only regulates differentiation and apoptosis, but it also exhibits a non-canonical structural role in poising chromatin for cleavage at an early stage of neutrophil cell death.

Association between the metabolic syndrome, its individual components, and unprovoked venous thromboembolism: results of a patient-level meta-analysis.

OBJECTIVE: The metabolic syndrome (MetS) may contribute to the pathogenesis of venous thromboembolism (VTE), but this association requires additional investigation. APPROACH AND RESULTS: We performed a patient-level meta-analysis of case-control and cohort studies that evaluated the role of MetS and risk of unprovoked VTE. For case-control studies, odds ratios and 95% confidence intervals were calculated using logistic regression analysis to estimate the influence of individual variables on the risk of VTE; χ(2) tests for trend were used to investigate the effect of increasing number of components of MetS on the risk of VTE and to explore the influence of abdominal obesity on this relationship. For cohort studies, hazard ratios and 95% confidence interval were calculated using multivariable Cox regression analysis. Six case-control studies were included (908 cases with unprovoked VTE and 1794 controls): in multivariate analysis, MetS was independently associated with VTE (odds ratio, 1.91; 95% confidence interval, 1.57-2.33), and both MetS and abdominal obesity were better predictors of unprovoked VTE than obesity defined by the body mass index. Two prospective cohort studies were included (26,531 subjects and 289 unprovoked VTE events): age, obesity, and abdominal obesity, but not MetS were associated with VTE. CONCLUSIONS: Case-control but not prospective cohort studies support an association between MetS and VTE. Abdominal adiposity is a strong risk factor for VTE.

α-Thalassemia does not seem to influence erythrocyte deformability in sickle cell trait carriers.

Studies dealing with rheological red blood cell (RBC) behavior in sickle cell trait carriers are scarce. Moreover, the association with α-thalassemia (α-thal), which also modifies erythrocyte behavior, has not always been taken into account. We analyzed erythrocyte deformability by means of a shear stress diffractometer, along with hematological and biochemical parameters (glucose and plasma lipids), given their possible influence on erythrocyte deformability, in 14 sickle cell trait carriers and 23 healthy controls. Nine patients were also α-thal carriers and five were not. Among the thalassemia carriers, eight were heterozygous and one was homozygous. When compared with controls, sickle cell trait carriers showed no differences for any of the biochemical parameters analyzed (p > 0.05), but significantly lower hemoglobin (Hb) (p = 0.003), mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) (p < 0.001) levels, although no differences in erythrocyte deformability were observed at any of the shear stresses tested (p > 0.05). When comparing sickle cell trait carriers, with and without α-thal, no differences in erythrocyte deformability were observed (p > 0.05), in spite of the former showing lower MCV and MCH (p < 0.05) levels. Carriers of α-thal had lower Hb S [ß6(A3)Glu → Val; HBB: c.20A > T] levels (p = 0.013) than non carriers. The existence of a compensating mechanism seems reasonable because, despite presenting lower erythrocyte indices, which could worsen erythrocyte deformability, this rheological property improves when the percentage of Hb S is lower.

Risk of venous thromboembolism associated with single and combined effects of Factor V Leiden, Prothrombin 20210A and Methylenetethraydrofolate reductase C677T: a meta-analysis involving over 11,000 cases and 21,000 controls.

Genetic and environmental factors interact in determining the risk of venous thromboembolism (VTE). The risk associated with the polymorphic variants G1691A of factor V (Factor V Leiden, FVL), G20210A of prothrombin (PT20210A) and C677T of methylentetrahydrofolate reductase (C677T MTHFR) genes has been investigated in many studies. We performed a pooled analysis of case-control and cohort studies investigating in adults the association between each variant and VTE, published on Pubmed, Embase or Google through January 2010. Authors of eligible papers, were invited to provide all available individual data for the pooling. The Odds Ratio (OR) for first VTE associated with each variant, individually and combined with the others, were calculated with a random effect model, in heterozygotes and homozygotes (dominant model for FVL and PT20210A; recessive for C677T MTHFR). We analysed 31 databases, including 11,239 cases and 21,521 controls. No significant association with VTE was found for homozygous C677T MTHFR (OR: 1.38; 95 % confidence intervals [CI]: 0.98-1.93), whereas the risk was increased in carriers of either heterozygous FVL or PT20210 (OR = 4.22; 95 % CI: 3.35-5.32; and OR = 2.79;95 % CI: 2.25-3.46, respectively), in double heterozygotes (OR = 3.42; 95 %CI 1.64-7.13), and in homozygous FVL or PT20210A (OR = 11.45; 95 %CI: 6.79-19.29; and OR: 6.74 (CI 95 % 2.19-20.72), respectively). The stratified analyses showed a stronger effect of FVL on individuals ≤ 45 years (p value for interaction = 0.036) and of PT20210A in women using oral contraceptives (p-value for interaction = 0.045). In this large pooled analysis, inclusive of large studies like MEGA, no effect was found for C677T MTHFR on VTE; FVL and PT20210A were confirmed to be moderate risk factors. Notably, double carriers of the two genetic variants produced an impact on VTE risk significantly increased but weaker than previously thought.

Mutations in the gene encoding the lamin B receptor produce an altered nuclear morphology in granulocytes (Pelger-Huët anomaly).

Pelger-Huët anomaly (PHA; OMIM *169400) is an autosomal dominant disorder characterized by abnormal nuclear shape and chromatin organization in blood granulocytes. Affected individuals show hypolobulated neutrophil nuclei with coarse chromatin. Presumed homozygous individuals have ovoid neutrophil nuclei, as well as varying degrees of developmental delay, epilepsy and skeletal abnormalities. Homozygous offspring in an extinct rabbit lineage showed severe chondrodystrophy, developmental anomalies and increased pre- and postnatal mortality. Here we show, by carrying out a genome-wide linkage scan, that PHA is linked to chromosome 1q41-43. We identified four splice-site, two frameshift and two nonsense mutations in LBR, encoding the lamin B receptor. The lamin B receptor (LBR), a member of the sterol reductase family, is evolutionarily conserved and integral to the inner nuclear membrane; it targets heterochromatin and lamins to the nuclear membrane. Lymphoblastoid cells from heterozygous individuals affected with PHA show reduced expression of the lamin B receptor, and cells homozygous with respect to PHA contain only trace amounts of it. We found that expression of the lamin B receptor affects neutrophil nuclear shape and chromatin distribution in a dose-dependent manner. Our findings have implications for understanding nuclear envelope-heterochromatin interactions, the pathogenesis of Pelger-like conditions in leukemia, infection and toxic drug reactions, and the evolution of neutrophil nuclear shape.

Hyperhomocysteinemia and the methylene tetrahydrofolate reductase C677T mutation in splanchnic vein thrombosis.

INTRODUCTION: The role that hyperhomocysteinemia (HH) and the C677T mutation in 5,10-methylenetetrahydrofolate reductase (MTHFR) play in splanchnic vein thrombosis (SVT) remains unclear due to this unusual thrombotic location. OBJECTIVE: To analyse the possible association of HH with the C677T mutation in the MTHFR gene in SVT. MATERIAL AND METHODS: We determined homocysteine levels and the C677T MTHFR mutation, along with classical cardiovascular risk factors, in 48 patients with SVT (18 Budd-Chiari syndrome, 11 mesenteric vein thrombosis, 19 portal vein thrombosis) and 84 controls. RESULTS: In the univariate analysis, patients with SVT showed statistically higher homocysteine levels (P =0.044). After adjusting for total cholesterol, differences disappeared (P =0.256). However, no differences in homocysteine levels were observed when comparing the three SVT types (P =0.199), even after adjusting for age and total cholesterol (P =0.095). In addition, the prevalence of the TT genotype was no different when controls were compared with patients with SVT (P =0.253) or with SVT subtypes (P =0.885). No association was found between HH (>15 µm) and the TT genotype in cases (P =0.404), controls (P =0.178), or in the different SVT subtypes (P =0.495). CONCLUSIONS: Our results suggest that HH and the homozygous genotype in the MTHFR C677T mutation do not seem to play a role in SVT development.

Parameters of inflammation in morbid obesity: lack of effect of moderate weight loss.

BACKGROUND: Obesity has been associated with a chronic activation of the acute-phase response. The aims of our study were to investigate whether levels of inflammatory cytokines are higher in obese patients, to evaluate their relationship with metabolic syndrome, and to analyze the effect of moderate weight loss upon their levels. METHODS: Sixty-seven severe or morbid obese patients were compared with 67 controls. Patients were submitted to a 4-week very low calorie diet followed by a low calorie diet for 2 months. Exclusion criteria were organic disease, ischemic heart disease or stroke, diabetes mellitus, hyperlipidemia, and hypertension. An evaluation was performed before and after the diet, in which fibrinogen, blood count, high-sensitive C-reactive protein (CRP), interleukin 6 (IL-6), and tumoral necrosis factor alpha (TNF-alpha) were measured. The Student t test was employed to compare differences between the groups and Pearson correlation coefficients were calculated. RESULTS: Obese patients showed higher levels of CRP (P < 0.001), IL-6 (P < 0.001), TNF-alpha (P < 0.001), leukocyte (P = 0.001), and neutrophil count (P < 0.001) than controls. In obese patients, inflammatory parameters were significantly correlated with anthropometric parameters and did not differ between obese subjects with or without metabolic syndrome. Moderate weight loss (excess weight loss 19.6%) was achieved through dieting, but no change was observed in any inflammatory parameter. CONCLUSIONS: Obesity is associated to a chronic inflammatory state that seems to be due to an increased secretion of cytokines, and this state is not related to the presence of metabolic syndrome. Moderate weight loss does not ameliorate this inflammatory state in the short term.

Activated protein C levels in obesity and weight loss influence.

Obesity is associated with a high risk of cardiovascular events. Several haemostatic disturbances which could contribute to this increased risk have been described in obesity; nevertheless, the state of coagulation inhibitors has been scarcely studied in these patients. The aim of the present study was to compare activated protein C levels in obese patients and in a control group, and to evaluate the effect of weight loss. In 67 severe or morbid obese patients, an evaluation was performed at baseline and 3 months after diet. The same determinations were performed in 67 healthy volunteers with normal body weight. We also quantified the levels of protein C and prothrombin fragment 1+2. Obese patients showed significantly higher levels of activated protein C, protein C and fragment 1+2. No correlation was found between activated protein C and fragment 1+2 levels in obese patients. After three months of diet, a significant decrease in activated protein C and fragment 1+2 was observed. In conclusion, activated protein C levels are increased in obese patients, but only a minor fraction of this increase may be explained by the higher thrombin generation and C protein levels. Activated protein C levels decrease with weight loss, due in part to a thrombin generation reduction.

Lack of association between hemorheological alterations and upper-extremity deep vein thrombosis.

INTRODUCTION: There is an association between rheological alterations and lower extremity deep vein thrombosis, although no research has been carried out regarding the possible influence of rheological alterations in the development of venous thrombosis in the upper limbs. MATERIALS AND METHODS: We have determined in 54 patients (23 male, 31 female; aged 35+/-11 years) with primary upper extremity deep vein thrombosis (UEDVT), and in a well matched control group of 87 subjects (41 male, 46 female; aged 36+/-12 years) the rheological profile (blood viscosity, plasma viscosity, fibrinogen, erythrocyte aggregation, erythrocyte deformability) along with plasma lipids (total cholesterol and triglycerides) and body mass index (BMI). Thrombophilic defects (antithrombin, protein C, protein S deficiencies, factor V Leiden, prothrombin G20210A mutation and antiphospholipid antibodies) were also determined. RESULTS: No statistical differences were observed in any of the rheological parameters analyzed (p>0.05), as well as in plasma lipids and BMI (p>0.05) when cases and controls were compared. In investigating the associated factors, we found that more than 60% of events could be explained through effort (28%), thrombophilic defects (19%) and oral contraceptives (26%). Obesity and hyperlipidaemia were not associated with primary UEDVT. CONCLUSION: Hemorheological alterations do not seem to contribute from a pathogenic point of view to the development of thrombotic events in this infrequent venous location, which is in accordance both with the absence of cardiovascular risk factors and the high shear forces existing in the upper extremity.

Erythrocyte aggregability and AB0 blood groups.

It is not established whether there is an association between erythrocyte aggregation and AB0 blood type, as glycophorins carry sialic acid which is responsible for the negative erythrocyte surface charge and the antigenicity for AB0 blood groups. We have determined erythrocyte aggregation by means of the Myrenne aggregometer in 114 healthy volunteers, along with plasma lipids, fibrinogen and AB0 blood groups. No differences in erythrocyte aggregation (EA0 and EA1) were observed when subjects with 0 (n = 45) and non-0 (n=69) blood group were compared (P = 0.624 and P = 0.838, respectively). Fibrinogen was statistically lower in 0 group compared with non-0 group (P = 0.012). Erythrocyte aggregation (EA0 and EA1) correlated significantly with both lipids and fibrinogen (P < 0.01). When erythrocyte aggregation was dichotomized as EA1 > or = 8, no association was found with 0 and non-0 blood groups (P > 0.05) but it was associated with high lipid levels: T-Chol > 220 mg/dl, TG > 175 mg/dl and high fibrinogen levels > 300 mg/dl (P = 0.035; P = 0.030; P = 0.010, respectively). Erythrocyte aggregation does not seem to be associated with blood groups, but rather with plasma lipids and fibrinogen.

Yasmin and venous thromboembolism: new case reports.

It is not yet known whether Yasmin involves a higher thrombotic risk compared with other contraceptives. We present a serie of eight new cases of women who developed thrombotic events early after starting on Yasmin who were sent to our Thrombosis and Hemostasis Unit for a thrombophilia work-up in the last five years. Only two of them were heterozygous carriers of the prothrombin G20210A mutation and three were obese while none of them were smoker. These new cases provide information about the characteristics of the thrombotic events and the concomitant risk factors, indicating that this pill may not be as safe as had been previously thought, and suggest that new studies regarding safety profile of Yasmin are required to explain the association with venous thrombotic events.

Identification of 58 Mutations (26 Novel) in 94 of 109 Symptomatic Spanish Probands with Protein C Deficiency.

Presently, no data on the molecular basis of hereditary protein C (PC) deficiency in Spain is available. We analyzed the PC gene (PROC) in 109 patients with symptomatic PC deficiency and in 342 relatives by sequencing the 9 PROC exons and their flanking intron regions. In 93 probands, we found 58 different mutations (26 novel). Thirty-seven consisted of a nucleotide change, mainly missense mutations, 1 was a 6-nucleotide insertion causing the duplication of 2 amino acids, and 4 were deletions of 1, 3, 4, and 16 nucleotides. Nine mutations caused type II deficiencies, with the presence of normal antigen levels but reduced anticoagulant activity. Using a PC level of 70% as lowest normal limit, we found no mutations in 16 probands and 25 relatives with PC levels ≤ 70%. On the contrary, 4 probands and 12 relatives with PC levels > 70% carried the mutation identified in the proband. The spectrum of recurrent mutations in Spain is different from that found in the Netherlands, where the most frequent mutations were p.Gln174* and p.Arg272Cys, and is more similar to that found in France, where the most frequent were p.Arg220Gln and p.Pro210Leu. In our study, p.Val339Met (9 families), p.Tyr166Cys (7), p.Arg220Gln (6), and p.Glu58Lys (5) were the most prevalent. This study confirms the considerable heterogeneity of the genetic abnormality in PC deficiencies, and allowed genetic counseling to those individuals whose PC levels were close to the lower limit of the normal reference range.

Fibrinolytic inhibitor levels and polymorphisms in Behçet disease and their association with thrombosis.

This study aimed to assess the fibrinolytic inhibitors and their association with thrombosis in Behçet disease. Thrombin activatable fibrinolysis inhibitor (TAFI) (P < 0.001) and plasminogen activator inhibitor-1 (PAI-1) levels (P = 0.022) were significantly higher in 79 patients than in 84 controls. No significant differences were observed in CPB2 (TAFI) Thr325Ile and SERPINE1 (PAI1) 4G/5G polymorphism distribution between patients and controls. TAFI activity levels were significantly higher in patients with thrombosis than in those without thrombosis (P = 0.024). In conclusion, the increased TAFI levels in Behçet disease could contribute to the increased risk of thrombosis observed in these patients.

Haplotypes of the EPCR gene, prothrombin levels, and the risk of venous thrombosis in carriers of the prothrombin G20210A mutation.

BACKGROUND: Haplotypes A1 and A3 in the endothelial protein C receptor (EPCR) gene are tagged by 4678G/C and 4600A/G respectively. We assessed whether these haplotypes modify the risk of venous thromboembolism in carriers of the prothrombin 20210A allele. DESIGN AND METHODS: We genotyped 4678G/C and 4600A/G in 246 20210A carriers: 84 venous thromboembolism propositi and 162 relatives (13 symptomatic), and in 140 relatives not carrying the 20210A variant. Prothrombin and soluble EPCR (sEPCR) levels were also measured. RESULTS: Among propositi, the mean age at first onset was lower in carriers (35 +/- 8 years) than non-carriers of the 4600G allele (44 +/- 14 years) (p = 0.004). The probability of being free of thrombosis at age 40 was lower in 20210A carriers with the EPCR 4600G allele (p = 0.015). The frequency of the 4600G allele (p=0.002) and the levels of prothrombin antigen (p = 0.002) and sEPCR (p < 0.001) were higher in the propositi than in their asymptomatic relatives. Multivariate analyses showed that the presence of the 4600G allele (OR = 2.5, 95% confidence interval 1.3-5.0), sEPCR > 147 ng/mL (2.8, 1.5-5.2) and prothrombin > 129% (3.8, 1.8-8.3) all increased the thrombotic risk. In bivariate analysis, including the 4600G allele and sEPCR > 147 ng/mL, only the latter remained associated with risk. CONCLUSIONS: These results show that in 20210A carriers the venous thromboembolism risk is influenced both by the actual prothrombin levels and by the EPCR A3 haplotype, via its effect on sEPCR levels.

Upper-extremity deep vein thrombosis in a patient on clozapine therapy carrying the prothrombin G20210A mutation.

Clozapine treatment for resistant schizophrenic disorders has been associated to venous thromboembolic events. We report the case of a patient who developed upper-extremity deep vein thrombosis just 2 months after starting on clozapine in whom the thrombophilia work-up revealed the presence of the prothrombin G20210A mutation.

Obesity and activated protein C resistance.

It has been reported that obesity may be associated with activated protein C resistance, which could increase the thrombotic risk in these patients. The aim of our study was to evaluate this parameter in obese patients and controls, as well as the effect of weight loss on this parameter. In 63 severely or morbidly obese patients and in 65 healthy volunteers, an anthropometric and analytical evaluation (activated protein C resistance and prothrombin fragment F1 + 2) was performed at baseline and after 3 months of diet. Obese patients showed higher levels of F1 + 2 than controls, whereas activated protein C sensitivity ratios showed no differences. After weight loss, prothrombin fragment F1 + 2 was reduced, but no differences were found in activated protein C sensitivity. We did not find an activated protein C-resistant phenotype in obese subjects.

Plasminogen activator inhibitor-1 levels in severe and morbid obesity. Effect of weight loss and influence of 4G/5G polymorphism.

INTRODUCTION: An association between an increase in plasminogen activator inhibitor type 1 and obesity has been described. It has also been shown that a decrease in adiposity has beneficial effects. However, less information is available regarding morbid obesity and hypofibrinolysis. The aim of the present study was to evaluate the effect of weight loss and the influence of the plasminogen activator inhibitor type 1 promoter 4G/5G genotype on plasminogen activator inhibitor type 1 levels in severe and morbid obesity. MATERIALS AND METHODS: Sixty-seven obese patients were studied before and three months after a weight reduction program, and compared with 67 controls. We determined plasminogen activator inhibitor type 1 antigen and activity levels, tissue type plasminogen activator antigen levels, 4G/5G genotype and biochemical parameters in both groups. RESULTS: A significant increase in plasminogen activator inhibitor type 1 antigen and activity was observed in obese patients in comparison with the control group (P<0.001). No significant differences in plasminogen activator inhibitor type 1 levels among 4G/5G genotypes were obtained. After weight loss, a significant decrease in plasminogen activator inhibitor type 1 antigen and activity was observed (P<0.001). A significant and positive correlation was observed in percentage changes in plasminogen activator inhibitor type 1 and body mass index (P=0.02). CONCLUSIONS: A decrease in body mass index in severe and morbid obesity shows a favourable effect on the fibrinolytic system due to a decrease in plasminogen activator inhibitor type 1 levels. However, no influence of 4G/5G polymorphism has been observed in this setting.

Haemorheological profile in patients with systemic sclerosis.

The relationship between rheological alterations and systemic sclerosis (SSc) is not well established. We have determined in 27 patients with SSc (4 male, 21 female ) aged 59 +/- 14 years and in a well matched control group the whole rheological profile, i.e. blood viscosity (BV), plasma viscosity (PV), erythrocyte aggregation (EA), erythrocyte deformability (ED) along with fibrinogen (Fbg), C-reactive protein (CRP), lipids, and erythrocyte indices. Patients show higher Fbg, PV and EA (P<0.01) and lower ED (P<0.01). A negative significant correlation was found between ED and inflammation markers, both CRP (P<0.05) and Fbg (P<0.01), indicating that decreased ED seems to be related to inflammatory changes at microcirculatory levels. In addition, patients with anticentromere antibodies show significantly lower ED than those without (P<0.05). The clinical significance of this observation needs to be clarified, deserving further research.

Haemorheological changes in patients with systemic lupus erythematosus do not seem to be related to thrombotic events.

Atherothrombotic events are significant factors of mortality and morbidity in patients with systemic lupus erythematosus (SLE). The extent that rheological factors may be involved in these events in these patients has not been established. We measured the following rheological parameters in 86 patients with SLE, of whom 16 had suffered venous and/or arterial thrombotic events, and in 86 healthy controls: fibrinogen (Fbg), plasma viscosity (PV), blood viscosity at 230 s(-1) both at native haematocrit (BVn 230 s(-1) and corrected to 45% (BVc 230 s(-1), erythrocyte aggregation at stasis (AE0) and at 3 s(-1) (AE1), aggregation time (Ta), aggregation index at 10 s (AI10), disaggregation threshold (gammaD), and erythrocyte deformability (ED). In addition glucose, total cholesterol (T-Chol), triglycerides (TG), haematocrit (Hct) and Body Mass Index (BMI) were determined. SLE Disease Activity Index (SLEDAI) was also assessed. The patients showed a significant increase in BMI (P=0.030), TG (P<0.001), PV (P=0.007), AE0 (P=0.005), AE1 (P=0.006), AI10 (P=0.024), gammaD (P=0.001), Fbg (P=0.050); and a significant decrease in Ta (P<0.001), Hct (P<0.001) and BVn 230 s(-1) (P=0.003). When patients with SLEDAI10 were compared, the latter had lower Hct (P=0.041) and lower BVn 230 s(-1) (P=0.017) than those with less SLEDAI. No significant differences were found in any of the parameters analysed on comparing patients who had suffered a thrombotic event with those who had not. Our results suggest that, although patients with SLE have moderate rheological changes, these do not seem to be responsible for the increase in the thrombotic tendency in these patients.