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.

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 of erythrocyte deformability with red blood cell distribution width in metabolic diseases and thalassemia trait.

Increased red blood distribution width (RDW) in anemia is related to disturbances in the cellular surface/volume ratio, usually accompanied by morphological alterations, while it has been shown in inflammatory diseases that the activity of pro-inflammatory cytokines disturbing erythropoiesis increases RDW. Recently it has been reported that higher RDW is related with decreased erythrocyte deformability, and that it could be related with the association of RDW and increased risk of cardiovascular diseases. In order to analyze the influence of morphological alterations and proinflammatory status on the relationship between RDW and erythrocyte deformability, we analyzed erythrocyte deformability along with RDW and other hematological and biochemical parameters in 36 α-thalassemia, 20 ß-thalassemia, 20 δß-thalassemia trait carriers, 61 metabolic syndrome patients and 76 morbidly obese patients. RDW correlated inversely with erythrocyte deformability in minor ß-thalassemia (r =-0.530, p <  0.05), and directly in both metabolic syndrome and morbidly obese patients (ρ= 0.270, p <  0.05 and ρ= 0.258, p <  0.05, respectively). Minor ß-thalassemia is often accompanied by more marked cell-shaped perturbations than other thalassemia traits. This could be the reason for this negative association only in this setting. Higher anisocytosis seems to be associated with greater morphologic alterations (shape/volume), which reduce erythrocyte deformability. The proinflammatory profile in metabolic patients can be related to the positive association of RDW with erythrocyte deformability found in these patients. However, further research is needed to explain the mechanisms underlying this association.

Red blood cell distribution width in patients with cryptogenic stroke.

BACKGROUND: There is no information about a possible association of red blood cell distribution width (RDW) with cryptogenic stroke (CS). We aimed to analyze the association of RDW with CS. PATIENTS AND METHODS: One hundred and sixty-three patients with CS were included along with 186 healthy controls. Fibrinogen, leukocytes, hemoglobin, and erythrocyte indices were evaluated. RESULTS: Patients showed higher RDW, leukocyte count, and body mass index (BMI) than controls (P < .05). No differences were observed in the erythrocyte indices or in glucose, cholesterol, and triglycerides levels (P > .05). When patients with anemia were excluded from the study (6 controls and 5 cases), the differences between cases and controls persisted (P = .005). Multivariate logistic regression revealed that, after adjusting for potential confounders (anemia, age > 40 years, gender, and fibrinogen >382 mg/dL, total cholesterol >240 mg/dL, and BMI > 28.7 kg/m(2)), RDW >14% was the only parameter that independently increased the risk of CS. CONCLUSION: The RDW >14% increased the risk of CS by 2.5-fold, irrespectively of anemia, inflammation, and lipidic profile.

Association of metabolic syndrome and its components with hyperuricemia in a Mediterranean population.

Several studies have found an association between hyperuricemia and metabolic syndrome (MS), although there are discrepancies as to which MS components play a pivotal role in this association. We aimed to investigate the association between serum uric acid (SUA) levels and MS in a Mediterranean population (eastern Spain). We performed a case-control study of 71 patients with MS and 122 healthy controls. MS was defined according to the revised National Cholesterol Education Program Adult Treatment Panel III modified criteria. Hyperuricemia was defined as SUA levels >6.55 mg/dL. We determined biochemical, lipidic and inflammatory parameters along with uric acid. Patients with MS showed a higher risk of hyperuricemia than those without MS (OR: 2.87 95% CI: 1.48- 5.55; p = 0.002). In turn, the unadjusted logistic regression analysis showed that hyperuricemia is associated with a higher risk of presenting all the MS components, except hypertension; i.e., hypertriglyceridemia, low HDL-cholesterol, abdominal obesity and glucose intolerance were predictors for hyperuricemia (OR: 3.15, 95% CI: 1.61- 6.15, p = 0.001; OR: 4.07, 95% CI: 1.77- 9.33, p = 0.001; OR: 2.81, 95% CI: 1.41- 5.58, p = 0.003 and OR: 2.82, 95% CI: 1.46- 5.45, p = 0.002 respectively). The adjusted logistic regression analysis revealed that only low HDL-cholesterol and glucose intolerance were independent predictors for hyperuricemia (OR: 2.71, 95% CI 1.06- 6.97, p = 0.038; OR: 2.14, 95% CI 1.01- 4.56, p = 0.049, respectively). In our geographical area, the patients with MS showed a nearly 3-fold risk of hyperuricemia than those without. Among all the MS components, low-HDL-cholesterol and high glucose independently increased more than twice the risk of hyperuricemia, and are the pivotal components involved in hyperuricemia.

Inflammatory markers and Lp(a) levels as cardiovascular risk factors in androgenetic alopecia.

It is not well-established whether patients with androgenetic alopecia (AGA) show a higher cardiovascular risk and higher prevalence of metabolic syndrome (MS). Therefore, we aimed to analyze the cardiovascular risk and the prevalence of MS by means of a case-control study. We determined lipidic, inflammatory, hormonal and insulin resistance parameters with conventional laboratory methods in 50 male early-onset AGA patients and 50 controls. AGA patients did not show statistical differences for insulin resistance (glucose, insulin, C peptide, HOMA), lipids (total-cholesterol, HDL-cholesterol, tryglicerides) or hormonal parameters (testosterone, free androgen index, sex hormone-binding globulin) P >  0.05, respectively. No differences between groups were observed in prevalence of MS or its components (P >  0.05). AGA patients showed higher levels of fibrinogen, C-reactive protein (CRP) and lipoprotein(a) (Lp(a)) (P = 0.016, P = 0.019 and P = 0.032, respectively). In the unadjusted logistic regression analyses, PCR >4 mg/L, fibrinogen >395 mg/dL and Lp(a) >59 mg/dL increased the risk of AGA, but in the adjusted logistic regression analyses, only PCR >4 mg/L and Lp(a) >59 mg/dL independently increased this risk (OR = 5.83, 95% CI 1.33-25.59 P = 0.020; OR = 3.94 CI 95% 1.08-14.43 P = 0.038). The present study indicates that AGA patients do not show differences in either insulin resistance or prevalence of MS. However, AGA patients show a higher cardiovascular risk characterised by an increase in inflammatory parameters and Lp(a) levels.

Increased circulating endothelial cells and microparticles in patients with psoriasis.

INTRODUCTION: Psoriasis is a chronic pathology characterized by increased inflammation that can be associated with changes in the vascular endothelium. We quantified the levels of circulating endothelial cells (CECs) and microparticles (MPs) in patients with psoriasis in order to analyze their relationship with endothelial and inflammation markers, subclinical atherosclerosis and microcirculation. METHODS: We studied 20 patients and 20 controls. Circulating markers of endothelial damage (CEC, MPs and von Willebrand factor, vWF) and inflammation (E-selectin, E-sel; Interleukin-6, IL-6 and C-reactive protein, CRP) were determined. Subclinical atherosclerosis was assessed by carotid ultrasound to obtain intima-media thickness. Microcirculation was evaluated by nailfold capillaroscopy. RESULTS: CECs, MPs, vWF, CRP and E-sel levels were significantly elevated in patients when compared with controls (p <  0.05). Ninety-four and fifty-three percentage of patients had CEC and MP levels higher than 99th percentile in controls. Forty-seven percent of patients simultaneously showed increased CEC and MP levels. MPs correlate with the inflammatory markers and with the intima-media thickness. CECs correlate with the capillaries loops per mm (p <  0.05). CONCLUSION: Psoriasis patients show elevated CECs and MPs, as a sign of endothelial dysfunction, which correlates with inflammatory markers as well as subclinical atherosclerosis and some capillaroscopy findings.

Red blood cell distribution width and erythrocyte deformability in patients with acute myocardial infarction.

Increased RDW has been found to be a marker of adverse outcomes in cardiovascular disease (CVD), although the exact mechanism remains unclear. Recently, several authors have found that higher RDW is associated with decreased erythrocyte deformability, which can impair blood flow through microcirculation, a fact which may explain the increased risk for CVD events associated with elevated RDW. The aim of the present study was to investigate the association between RDW and erythrocyte deformability in patients with acute myocardial infarction (AMI). The study group comprised 60 AMI patients and 72 gender- and age-matched controls, in whom erythrocyte deformability was determined by means of the elongation index (EI) in a Rheodyn SSD, along with haematological, biochemical and inflammatory parameters. Patients showed higher RDW (p = 0.012) and lower EI (p < 0.05) than controls. When anaemic patients were removed from the study, AMI showed still lower EI than controls (p < 0.05), but no differences in RDW were observed (p = 0.141). RDW correlated inversely with haematimetric indices (p < 0.001), but not with inflammatory and biochemical parameters (p > 0.05). EI correlated inversely with Hb, MCHC (p < 0.001) and directly with MCV (p < 0.05). EI also correlated inversely with glucose (p < 0.05) and directly with HDL-cholesterol (p < 0.05). The multivariate regression model showed that only MCV and Hb were independent predictors of RDW (beta coefficients: -0.383, -0.208; p < 0.001, p = 0.050, respectively). In addition, MCV, MCHC and hyperlipidaemia were independent predictors of EI (beta coefficients: 0.366, -0.533, -0.192; p < 0.001, p < 0.001, p = 0.019 respectively). In AMI patients, increased RDW is not related with EI, so this mechanism does not seem to be responsible for an increased CDV risk in these patients.

Influence of inflammatory and lipidic parameters on red blood cell distribution width in a healthy population.

Red blood cell distribution width (RDW) is a routine red blood cell count parameter which has been shown to be associated with inflammatory parameters. Recently, some authors proposed that RDW seems to be a marker of an adverse lipidic profile. In order to clarify whether RDW is related to inflammation, plasma lipids, or both, we determined anthropometric, hematimetric, inflammatory and lipidic parameters in 1111 healthy subjects. RDW correlated directly with age, body mass index (BMI), inflammatory parameters (plasma viscosity, erythrocyte sedimentation rate (ESR), fibrinogen, leukocyte and neutrophil count), and inversely with iron and hematimetric parameters (P <  0.05). When subjects were divided according to gender, RDW correlated inversely with triglycerides only in women (P <  0.05). When subjects were classified into RDW-quartiles, increased RDW values were accompanied by decreased serum iron levels and hematimetric indices (P <  0.01), whereas age and inflammatory markers increased according to RDW-quartiles (P <  0.001 and P <  0.05, respectively). However, plasma lipids did not change with increasing RDW-quartiles (P >  0.05). In the linear regression analysis, age, hemoglobin, MCV (beta coefficient: 0.202, -0.234, -0.316, P <  0.001) and fibrinogen (beta coefficient: 0.059, P = 0.048) were the only independent predictors of RDW. The present study indicates that RDW is associated with inflammatory markers and hematimetric indices, but not with plasma lipid levels in a healthy population.

Subclinical hypothyroidism and cardiovascular risk.

BACKGROUND: Subclinical hypothyroidism (SCH) has been suggested to be associated with increased cardiovascular risk by different mechanisms. Several cardiovascular risk factors have been analysed, but yielded controversial results. OBJECTIVES: We aimed to analyse whether there are differences in several cardiovascular risk markers, such as lipids, inflammatory parameters: plasma viscosity (PV), fibrinogen and C reactive protein (CRP); homocysteine (Hcy) and red blood cell distribution width (RDW), when comparing SCH and controls. We also analysed which of these parameters predict SCH risk and constitute independent markers. METHODS: We determined PV in a Fresenius capillary plasma viscosimeter, Hcy by a chemiluminiscent enzyme immunoassay, and biochemical and haematological parameters by conventional laboratory methods in 58 SCH outpatients and 58 controls matched for age and gender. RESULTS: SCH patients did not show statistical differences for glucose, lipids or leucocytes (p > 0.05). However, patients showed a higher prevalence for use of hypolipidaemic drugs, body mass index (BMI), thyroid stimulating hormone (TSH), PV, CRP, fibrinogen, Hcy and RDW (p < 0.05). RDW correlated with inflammation parameters: PV (r = 0.331, p < 0.05), fibrinogen (r = 0.424, p < 0.05), CRP (r = 0.433, p < 0.01) and leucocytes (r = 0.613, p < 0.01). None of the cardiovascular markers correlated with the TSH levels (p > 0.05) In the unadjusted logistic regression analyses, BMI ≥28 kg/m2, RDW ≥14%, Hcy ≥12 µm/L, fibrinogen ≥400 mg/dL and MCV ≤88 fL increased SCH risk, but only RDW ≥14% and fibrinogen ≥400 mg/dL independently increased this risk in the adjusted logistic regression analyses (OR = 4.68, 95% CI 1.20-18.30 P = 0.026; OR = 3.48, 95% CI 1.08-11.23 P = 0.037). CONCLUSION: SCH patients show a higher cardiovascular risk, characterised by increased PV, fibrinogen, Hcy and RDW. However, only fibrinogen ≥400 mg/dL and RDW ≥14% are independent predictors of SCH.

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.

Red blood cell distribution width is not related with inflammatory parameters in morbidly obese patients.

OBJECTIVE: Red blood cell distribution width (RDW) is a hematological parameter that has been studied in several clinical settings and has been found to be related to both anemia and inflammatory status. As obesity is related to increased inflammatory pattern, we aimed to analyze the RDW in this setting. METHODS: We determined hematological and inflammatory parameters in morbidly obese patients before bariatric surgery (n=142) and normo-weight controls (n=144). RESULTS: RDW was higher in patients than in controls (p<0.001), along with C-reactive protein (p<0.001) and fibrinogen, (p<0.001) while hemoglobin (p=0.026), serum iron (p<0.001), MCH (p=0.002) and MCHC (p<0.001) were lower in morbidly obese patients. The logistic correlation analysis revealed that only low serum iron (<62 µg/dL) and MCH (<28.14 pg) levels were associated with RDW>14% (OR 7.61, 95% CI: 1.93-30.04, p=0.004; OR 5.67, 95% CI: 1.98-16.24, p=0.001; respectively). CONCLUSIONS: These data indicate that the elevated RDW in morbidly obese patients reflects a mild red blood cell hypochromia that does not relate to inflammatory parameters, but to hyposideremia and, consequently, to lower erythrocyte indices, possibly as a result of being on a very low-calorie diet before bariatric surgery. Therefore, RDW should not be considered as an inflammatory marker in this clinical setting.