The effect of endothelial Nitric Oxide Synthase G894T and T786C polymorphisms on Hypoxia-Inducible Factor-1 alpha expression in Sickle Cell Disease.

Hypoxia-Inducible Factor-1α (HIF-1α) expression is upregulated in Sickle Cell Disease (SCD) and correlates with various laboratory markers of disease severity. Nitric Oxide plays a pivotal role in SCD pathophysiology and endothelial Nitric Oxide Synthase (NOS3) polymorphisms affect prognosis and laboratory parameters. This study questions the effect of NOS3 G894T and T786C polymorphisms on HIF-1α expression in SCD. We show that G894T polymorphism is a significant predictor of HIF-1α expression. Its effect is exerted independently of hemolysis/hemoglobin fragment concentrations, as shown in multiple regression analysis. Our results establish a novel modulator of HIF-1α expression on the mRNA level and indirectly support the role of nitric oxide in the pathophysiology of SCD.

Reduced peripheral blood superoxide dismutase 2 expression in sickle cell disease.

Sickle cell disease (SCD), a hereditary form of chronic hemolytic anemia, is characterized by acute vascular occlusion and chronic complications as pulmonary hypertension (PH), a hallmark of higher mortality. This study aimed to determine peripheral blood expression of superoxide dismutase 2 (SOD2), a major mitochondrial antioxidant enzyme in SCD patients on the mRNA level and compared it with SOD2 expression in healthy individuals. It also aimed to detect possible differences in SOD2 expression among patients with/without specific SCD complications and to detect possible correlations with patient laboratory parameters. SOD2 mRNA levels were significantly lower in SCD patients in comparison with controls and correlated with red blood cell count, reticulocyte count, platelet count, C-reactive protein, ferritin, and brain natriuretic peptide values. SCD patients with echocardiographic indications of PH featured significantly reduced SOD2 expression in comparison with patients without such indications. Consequently, SOD2 expression emerges as a potential biomarker of PH in SCD being a link among hemolysis, inflammation, iron overload, oxidative stress, and SCD cardiopathy.

Data on eNOS T786 and G894T polymorphisms and peripheral blood eNOS mRNA levels in Sickle Cell Disease.

In this article, we present data on endothelial Nitric Oxide Synthase (eNOS) gene T786C and G894T polymorphisms in Greek steady-state Sickle Cell Disease patients in comparison to healthy controls. Moreover, eNOS mRNA levels were determined in peripheral blood samples from 18 patients and 9 controls. This article complements our recently published article named "Prognostic value of eNOS T786C and G894T polymorphisms in Sickle Cell Disease" (I. Armenis, V. Kalotychou, R. Tzanetea, Z. Kontogeorgiou, D. Anastasopoulou, M. Mantzourani, M. Samarkos, K. Pantos, K. Konstantopoulos, I. Rombos, 2016) [1].

Prognostic value of T786C and G894T eNOS polymorphisms in sickle cell disease.

Endothelial Nitric Oxide Synthase (eNOS) is crucial for vascular homeostasis. Polymorphisms T786C and G894T affect eNOS regulation and have been related to various diseases. Sickle Cell Disease (SCD), a clinically diverse chronic hemolytic anemia, implies impaired nitric oxide bioavailability. Our aim was to determine eNOS genotype for T786C and G894T polymorphisms in Greek patients with SCD and to elucidate its consequences and effects if any on clinical phenotype. Seventy nine steady state cases, mostly compound heterozygous for Sickle Cell anemia/beta thalassemia and 48 controls were measured. Peripheral blood DNA was extracted and genotyped with PCR-RFLPs and Sanger sequencing. Total RNA was extracted from 18 patients and 9 controls and eNOS mRNA levels were determined by real-time PCR. Genotypes, allele distribution and eNOS mRNA levels did not differ between patients and controls, or among patients with different beta globin gene mutations. The 786CC genotype was more common in S/S and ß0/S patients with retinopathy. Moreover, 894TT S/S and ß0/S patients tended to have a higher hematocrit than 894GG and GT ones. However, the T786C eNOS genotype does not seem to affect peripheral blood cell-derived eNOS mRNA levels, at least in steady state conditions. This work is the first one describing the effects of eNOS polymorphisms on different forms of SCD, the first enrolling SCD patients of Caucasian origin and the first determining eNOS mRNA levels in peripheral blood from steady-state SCD patients.

Polymorphisms of uridine glucuronosyltransferase gene and irinotecan toxicity: low dose does not protect from toxicity.

Uridine glucuronosyltransferase (UGT) gene polymorphisms have been linked to irinotecan toxicity. Our purpose was to study the association between UGT1A1*28, UGT1A7*2, and UGT1A7*3 polymorphisms and irinotecan toxicity in Greek patients receiving low-dose weekly irinotecan. Blood samples were collected for 46 patients. DNA was extracted and UGT1A1 promoter and UGT1A7 exon 1 genotyping was carried out. Laboratory tests and physical examination were performed on regular basis for the assessment of toxicity. UGT1A1*28 was significantly correlated with both haematologic and non-haematologic toxicity. Moreover, patients carrying UGT1A7 polymorphisms had significant incidence of toxicity. To conclude, UGT polymorphisms play a role in the toxicity of irinotecan, even if the drug is administered in low doses. The genotyping test may be a useful tool for the management of patients who are going to receive irinotecan.

Adhesion molecules and high-sensitivity C-reactive protein levels in patients with sickle cell beta-thalassaemia.

BACKGROUND AND AIM: The primary symptoms of sickle cell disease (SCD) arise from vaso-occlusive crises. The pathogenesis of these crises is complex phenomenon where endothelial activation and damage has a major role. Chronic inflammation also plays an important role in the pathophysiology of SCD. We aimed to investigate endothelial activation in Caucasian Greek patients with SCD by means of measuring adhesion molecules and markers of inflammation. SUBJECTS AND METHODS: Twenty-eight patients with SCD aged 5-63 years were included in the study. Most of the patients (23/28) were double heterozygotes for sickle cell/beta-thalassaemia, while five patients (5/28) were sickle cell homozygotes. Patients were treated with one/or more of hydroxyurea, therapeutic phlebotomies, blood transfusion or splenectomy. Twenty apparently healthy individuals matched for age and sex formed the control group. Measurements of soluble intercellular adhesion molecule-1, (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), P-selectin, E-selectin, soluble thrombomodulin (sTM) and high-sensitivity C-reactive protein (hs-CRP) levels were performed using immunoassays in both patients and healthy individuals. RESULTS: We found that all endothelial adhesion molecules and hs-CRP were significantly increased (P < 0·001) in patients with SCD compared with controls, while sTM levels did not differ significantly (P > 0·05) and this increase was not influenced by the treatment. CONCLUSION: Our findings demonstrate the high degree of endothelial activation and damage seen in sickle cell patients even in steady-state condition, as well as the important chronic inflammation underlying the pathophysiology of this widespread disease.

Contribution of G71R mutation to Gilbert's syndrome phenotype in a Greek patient: A case report.

Gilbert's syndrome is characterized by a benign indirect hyperbilirubinemia. It has often been underestimated and undiagnosed because of its mild symptoms; although it is not as rare as was once believed when its frequency was estimated using data originating from biochemical tests. Based on molecular techniques, the occurrence of Gilbert's syndrome has changed, increasing to 10% in the Caucasian population. This molecular defect was described, by Bosma et al, in 1995, and affects the promoter region of the UGT 1A1 gene. In this case report, our aim is to present a new combination of two molecular defects in a Greek patient with Gilbert's syndrome. A 13-year-old Greek girl was examined for Gilbert's syndrome using molecular techniques, and an uncommon genotype was revealed comprising the rare mutation G71R in trans with A(TA)7TAA motif. The G71R mutation according to the literature, as well as our epidemiological data, is rare in Caucasians, while it is common in Asian populations. This is the first case study in the Greek population to report a new genotype for Gilbert's syndrome manifestation in the Caucasian population.

Erythrocytosis due to a combination of the high oxygen affinity hemoglobin variant, Hb Olympia [beta20(B2)Val-->Met] with beta- and alpha-thalassemia mutations: first case in the literature.

A 40-year-old Greek male was admitted to the hospital because of acute respiratory infection. The patient has been undergoing regular venesection for erythrocytosis for 20 years; he has also been taking oral anticoagulants for thrombosis for 15 years. The molecular defect for erythrocytosis was detected together with the rare Hb Olympia (HBB:c.61G>A) variant. This hemoglobin (Hb) variant was found in combination with two thalassemia-type globin gene defects, namely beta(0)-thalassemia (beta(0)-thal), HBB:c.118C>T and alpha(0)-thal (- -(MED)). This combination of three molecular defects is the first such case reported in the literature.

Analysis of the A(TA)(n)TAA configuration in the promoter region of the UGT1 A1 gene in Greek patients with thalassemia intermedia and sickle cell disease.

Gilbert's syndrome is characterized by mild unconjugated hyperbilirubinemia. The molecular basis of this syndrome usually concerns an additional dinucleotide insertion (TA) in the A(TA)(n)TAA configuration residing in the promoter region of the UGT1 A1 gene. This configuration may vary in length; the "n" represents the different number of TA repeats. The homozygosity A(TA)(7)TAA/A(TA)(7)TAA is involved in Gilbert's syndrome. In many cases of patients with thalassemia intermedia and sickle cell disease considerable variation in bilirubin levels is observed. In this study we investigated the contribution of the A(TA)(7)TAA/A(TA)(7)TAA genotype in the variable unconjugated serum bilirubin levels in 31 Greek patients with thalassemia intermedia and 27 Greek compound heterozygotes for beta thalassemia and sickle cell anemia. Analysis of the A(TA)(n)TAA configuration in the promoter region of the latter patients showed that those who were carrying the homozygosity A(TA)(7)TAA/A(TA)(7)TAA had higher levels of unconjugated bilirubin. These findings suggest that the coexistence of Gilbert's syndrome in patients with thalassemia intermedia and sickle cell disease may be the cause of the elevated values of unconjugated bilirubin, reducing the possibility of excessive hemolysis in these patients.

Plesiomonas shigelloides sepsis in a thalassemia intermedia patient.

Bacteremia due to Plesiomonas shigelloides was associated with rapidly fulminant septicemia, disseminated intravascular coagulation and massive adrenal hemorrhage in a splenectomized patient suffering from thalassemia intermedia who was treated with hydroxyurea. P. shigelloides was isolated in blood cultures; despite a vigorous combination of antibiotics the patient died after 24 h in the ICU. Lethal sepsis due to P. shigelloides has not previously been reported in Greece.

Amelioration of painful crises in sickle cell disease by venesections.

Sickle cell disease patients who acquire iron deficiency may experience a degree of amelioration from painful crises in terms of frequency, severity, and duration. This observation prompted us to identify the potential utility of iron load reduction in the management of this disease. Thirteen sickle cell patients not ameliorated by conventional treatment entered a weekly venesection protocol. Hematological values and painful crises of all degrees of severity were recorded and compared to those of the last 12 months before venesection for each case separately ("historical controls"). A decrease was noted in the frequency and intensity of several types of painful crises. Reduction of iron load by venesection seems to be a simple, safe, side-effect-free, and efficient way of preventing and ameliorating to a large extent painful crises in sickle cell disease. The biological effects of venesection on other parameters of sickle cell disease remain to be determined.