Genotoxic effect of iron overload and disease complications in transfused ß thalassaemic patients.

In previously reported studies, we observed significantly high genotoxicity biomarkers in regularly transfused thalassaemic patients, thus, in this study, we better investigated the genotoxic effect of iron overload and of thalassaemia complications, including their drug treatments. The assessment was performed in 64 regularly transfused thalassaemic patients using cytokinesis-block micronucleus and comet assays. All patients were splenectomised and undergoing iron chelation therapy. To reduce hypoxia-induced oxidative damage, the patients with haemoglobin levels <9.5 g/dL were excluded. Serum concentrations of ferritin, iron, transferrin and the percentage of transferrin saturation, as well as cardiac and hepatic T2* magnetic resonance imaging, were considered to evaluate serum and organ siderosis.All genotoxic biomarkers significantly differed between patients and healthy subjects. Iron intake via blood transfusions was inversely related to percentage of DNA in tail. The disease complications affecting endpoints were active Hepatitis C virus infection, drug therapy for osteoporosis (i.e. bisphosphonates) and hormone replacement therapy for hypogonadism.The results, highlighting the combined effect of iron overload and, mainly, disease complications, including their respective pharmacological treatments, confirmed the increased cancer risk in thalassaemic patients.

Soluble hemojuvelin in transfused and untransfused thalassaemic subjects.

OBJECTIVE: The hemojuvelin-bone morphogenetic protein axis is the principal iron-dependent mechanism of hepcidin regulation. The determination of soluble hemojuvelin (sHJV) levels could allow for a better understanding of the pathophysiological mechanisms of hepcidin regulation in thalassaemia. METHOD: We have assessed sHJV in 45 transfused and 15 untransfused thalassaemic patients in comparison with 15 healthy subjects, evaluating its relationships with some parameters of iron overload, anaemia and erythropoiesis. RESULTS: Untransfused thalassaemic patients had more severe anaemia and erythropoietic activity, while in transfused patients, the transfused RBCs reduced % reticulocytes and sTfR, increased serum indices of iron overload and iron stores in the liver (low MRI T2* values). sHJV levels were higher in patients than in controls and in untransfused in comparison with transfused patients. In the transfused group, we also found that sHJV values are significantly related to serum ferritin, cardiac MRI T2* and growth differentiation factor 15 and are sensitive to hepatitis C virus infection. CONCLUSION: These results suggest that sHJV synthesis seems to be affected by an erythropoietic/hypoxic signal in untransfused patients that have severe anaemia, while in regularly transfused subjects, it is influenced by iron stores.

The role of anaemia in oxidative and genotoxic damage in transfused ß-thalassaemic patients.

OBJECTIVES: Redox imbalance and genotoxic damage are commonly observed in ß thalassaemic patients. The aim of this study was to assess the role of anaemia in oxidative and genotoxic damage in regularly transfused thalassaemic patients, undergoing iron chelation therapy. METHODS: We studied the relationships of haematological, biochemical and clinical parameters with oxidative (reactive oxygen species and 8-oxo-7,8-dihydro-2'-deoxyguanosine) and genotoxic biomarkers (Comet assay and cytokinesis-block micronucleus test) in blood samples from 105 patients. To reduce the early effect of redox-active iron, samples were collected when pharmacokinetics of the iron chelators ensured their maximum effectiveness. The transfusion regimen, cardiac and hepatic magnetic resonance imaging T2* were evaluated to characterize the patient cohort. Labile plasma iron (LPI) was also assayed. RESULTS: Haemoglobin level had a significant effect on ROS, %DNA in the tail and micronuclei-micronucleated cell frequency (p < 0.05). Higher Hb values reduced redox imbalance. LPI, detectable in 50.5% of patients, was related to the number of apoptotic and necrotic lymphocytes (p = 0.03), demonstrating the cytotoxic effect of iron. DISCUSSION: The results highlight that an adequate transfusion regimen is essential to limit oxidative and genotoxic damage in ß-thalassemic patients undergoing chelation therapy. CONCLUSION: Owing to the higher risk of cancer in the thalassaemic cohorts, specific genotoxicity/oxidative biomarkers should be monitored in order to ameliorate and formulate more personalized disease management.

Effects of strontium ranelate on bone mass and bone turnover in women with thalassemia major-related osteoporosis.

Subjects affected by thalassemia major (TM) often have reduced bone mass and increased fracture risk. Strontium ranelate (SrR) is an effective treatment for postmenopausal and male osteoporosis. To date, no data exist on the use of SrR in the treatment of TM-related osteoporosis. Our aim was to evaluate the effects of SrR on bone mineral density (BMD), bone turnover markers and inhibitors of Wnt signaling (sclerostin and DKK-1). Twenty-four TM osteoporotic women were randomized to receive daily SrR 2 g or placebo in addition to calcium carbonate (1,000 mg) and vitamin D (800 IU). BMD at the lumbar spine and femoral neck, bone turnover markers (C-terminal telopeptide of procollagen type I [CTX], bone-specific alkaline phosphatase [BSAP]) and insulin-like growth factor-1 (IGF-1), sclerostin and DKK-1 were assessed at baseline and after 24 months. Back pain was measured by visual analog scale (VAS) every 6 months. After 24 months, TM women treated with SrR had increased their spine BMD values in comparison to baseline (p < 0.05). Moreover, they also exhibited a reduction of CTX and sclerostin levels (but not DKK-1) and exhibited an increase of BSAP and IGF-1 (p < 0.05); however, no significant changes were observed in the placebo group. In the SrR group, a reduction of back pain was observed after 18 months in comparison to baseline (p < 0.05) and after 24 months in comparison to placebo (p < 0.05). Our study reports for the first time the effects of SrR in the treatment of TM-related osteoporosis. SrR treatment improved BMD and normalized bone turnover markers, as well as lowering sclerostin serum levels.

Thalassaemia major and infectious risk: High Mobility Group Box-1 represents a novel diagnostic and prognostic biomarker.

High mobility group box -1 (HMGB1) represents a common causal agent for various types of diseases, including infective pathologies. This study aimed to investigate the role of HMGB1 in ß-thalassemia major (TM) by evaluating its diagnostic and prognostic role. Fifty-one TM patients and 30 healthy subjects (HS) were enrolled. Receiver operating characteristics (ROC) analysis was employed to calculate the area under the curve (AUC) for HMGB1 to determine the best cut-off values capable of identifying infectious episodes. Adjusted risk estimates for infective events were calculated using univariate followed by multivariate Cox proportional hazard regression analysis. Serum HMGB1 levels were higher in TM patients than in HS (14·6 ± 8·7 vs. 2·08 ± 0·9 ng/ml, P < 0·0001). Patients who underwent splenectomy were characterized by lower levels of HMGB1, when compared with patients with an intact spleen (10·2 ± 8 vs. 19·1 ± 7 ng/ml, P = 0·004). ROC analyses revealed an AUC for serum HMGB1 of 0·801, with a sensitivity and specificity of 92·3% and 68·2% to detect an infectious episode. Low HMGB1 levels predicted high risk of infective events (HR: 0·81; P = 0·006). HMGB1 represents a prognostic marker for TM patients and a predictive factor for infectious events.

Endocrinopathies, metabolic disorders, and iron overload in major and intermedia thalassemia: serum ferritin as diagnostic and predictive marker associated with liver and cardiac T2* MRI assessment.

INTRODUCTION: Endocrinopathies and metabolic disorders-characterized ß thalassemic (ßT) patients and the prevention and treatment of these comorbidities are important targets to be achieved. The aim of the study was to analyze the diagnostic and prognostic role of ferritin for endocrinopathies and metabolic disorders in ßT patients. The ability of iron chelators to treat iron overload and to prevent or reverse metabolic disorders and endocrinopathies was also evaluated. PATIENTS AND METHODS: Seventy-two ßT patients were treated with different chelation strategies during the study. Receiver operating characteristics analysis was employed to calculate the area under the curve for serum ferritin to find the best cutoff values capable of identifying endocrine dysfunction in thalassemic patients. Kaplan-Meier curves were generated to assess the incidence of endocrinopathy. Adjusted risk estimates for endocrinopathy were calculated using univariate followed by multivariate Cox proportional hazard regression analysis. RESULTS: High ferritin levels were observed in patients with hypothyroidism [1500 (872.5-2336.5) µg/L], hypogonadism [878 (334-2010) µg/L], and in patients with hypoparathyroidism or osteoporosis [834 (367-1857) µg/L]. A strict correlation between ferritin and T2* magnetic resonance imaging of heart (r = -0.64; P:0.0006) and liver (r = -0.40; P:0.03) values was observed. Patients with ferritin values above 1800 µg/L experienced a significantly faster evolution to hypothyroidism [log-rank (χ(2) ):7.7; P = 0.005], hypogonadism [log-rank (χ(2) ):10.7; P = 0.001], and multiple endocrinopathies [log-rank (χ(2) ):5.72; P = 0.02]. Ferritin predicted high risk of endocrine dysfunction independently of confounding factors (HR:1.23; P < 0.0001). The intensification of chelation therapy led to an amelioration of hypothyroidism. CONCLUSIONS: Ferritin represents a prognostic marker for ßT patients and a predictive factor for progression to endocrine dysfunctions. Intensive chelation therapy allows the reversibility of hypothyroidism.

Cardiac complications and diabetes in thalassaemia major: a large historical multicentre study.

The relationship between diabetes mellitus (DM) and cardiac complications has never been systematically studied in thalassaemia major (TM). We evaluated a large retrospective historical cohort of TM to determine whether DM is associated with a higher risk of heart complications. We compared 86 TM patients affected by DM with 709 TM patients without DM consecutively included in the Myocardial Iron Overload in Thalassaemia database where clinical/instrumental data are recorded from birth to the first cardiovascular magnetic resonance (CMR) exam. All of the cardiac events considered were developed after the DM diagnosis. In DM patients versus non-DM patients we found a significantly higher frequency of cardiac complications (46.5% vs. 16.9%, P < 0.0001), heart failure (HF) (30.2% vs. 11.7%, P < 0.0001), hyperkinetic arrhythmias (18.6% vs. 5.5%, P < 0.0001) and myocardial fibrosis assessed by late gadolinium enhancement (29.9% vs. 18.4%, P = 0.008). TM patients with DM had a significantly higher risk of cardiac complications [odds ratio (OR) 2.84, P < 0.0001], HF (OR 2.32, P = 0.003), hyperkinetic arrhythmias (OR 2.21, P = 0.023) and myocardial fibrosis (OR 1.91, P = 0.021), also adjusting for the absence of myocardial iron overload assessed by T2* CMR and for the covariates (age and/or endocrine co-morbidity). In conclusion, DM significantly increases the risk for cardiac complications, HF, hyperkinetic arrhythmias and myocardial fibrosis in TM patients.

Thyroid dysfunction in thalassaemic patients: ferritin as a prognostic marker and combined iron chelators as an ideal therapy.

OBJECTIVE: Endocrine complications characterised patients with ß thalassaemia (ßT). In particular, thyroid dysfunction occurs frequently in ßT major, but its long-term natural history is poorly understood. DESIGN: A total of 72 ßT patients were followed for 8 years. The incidence of thyreopathies, defined as the primary study endpoint, was assessed. The aim of this study was to analyse the prognostic role of ferritin for thyreopathies in patients with major and intermedia ßT. The power of different iron chelators to treat iron overload and to prevent or reverse thyreopathies was also assessed. METHODS: Patients were treated with chelators with different chelation strategies during the study. Receiver operating characteristics analysis was employed to calculate the area under the curve for serum ferritin to find the best cutoff values capable of identifying thyroid dysfunction in thalassaemic patients. Kaplan-Meier curves were generated to assess incidence of thyreopathy. Adjusted risk estimates for thyreopathy were calculated using univariate followed by multivariate Cox proportional hazard regression analysis. RESULTS: PATIENTS WITH THYROID DYSFUNCTION WERE CHARACTERISED BY HIGHER FERRITIN WHEN COMPARED WITH PATIENTS WITHOUT THYREOPATHIES (1500 (8722336) VS 513 (370698) G/L; P0.0001). PATIENTS WITH FERRITIN VALUES ABOVE 1800G/L EXPERIENCED A SIGNIFICANTLY FASTER EVOLUTION TO ENDPOINT (LOG-RANK ((2)): 7.7; P=0.005). Ferritin predicted high risk of thyroid dysfunction independently of confounding factors (hazard ratio: 1.20; P<0.0001). The intensification of chelation therapy led to an amelioration of thyroid function. CONCLUSIONS: Ferritin represents a prognostic marker for ßT patients and a predictive factor for progression to thyroid dysfunction. Intensive chelation therapy allows the prevention and reversibility of thyroid complications.

Early identification of cardiovascular involvement in patients with ß-thalassemia major.

The aim of the present study was to evaluate left ventricular myocardial deformation and carotid arterial stiffness using 2-dimensional strain and echo-tracking in patients with asymptomatic ß-thalassemia major (ß-TM) without significant myocardial iron overload to determine whether early subclinical cardiovascular abnormalities would be detectable. We enrolled 32 patients with ß-TM (23 women, mean age 35 ± 8 years) and 33 healthy volunteers (20 women, mean age 35 ± 6 years). All subjects underwent echocardiography with 2-dimensional strain analysis (XStrain) and ultrasonography of the carotid arteries with measurement of the stiffness parameters (ProSound Alpha 10). Cardiac magnetic resonance imaging using a T2* algorithm (37.7 ± 5.6 ms) for the assessment of myocardial iron overload was performed in each patient. The clinical and standard echocardiographic parameters were comparable between the patients and healthy subjects. The global left ventricular longitudinal strain was significantly impaired in the patients compared with the controls (-17.9 ± 3.5% vs -24.3 ± 3.4%, p = 0.002), although the radial and circumferential strain values were similar between the 2 groups (p = NS for both). The carotid intima-media thickness was comparable between the patients and healthy subjects (0.67 ± 0.20 mm vs 0.66 ± 0.15 mm, p = NS). In contrast, the arterial stiffness was significantly increased in the patients compared with the controls (stiffness index 6.16 ± 1.31 vs 4.65 ± 0.82, p <0.001; arterial compliance 1.10 ± 0.26 vs 1.28 ± 0.30 cm(2)/mm Hg, p = 0.027; elastic modulus 74.1 ± 19.5 vs 59.1 ± 12.1 mm Hg, p = 0.001). In conclusion, cardiovascular abnormalities, although often subclinical, occur at an early stage of ß-TM and also in the absence of significant iron overload. Thus, 2-dimensional strain and echo-tracking might be more accurate than standard echocardiography and vascular parameters in the early identification of cardiovascular involvement.

Oxidative damage and genotoxicity biomarkers in transfused and untransfused thalassemic subjects.

Chronic anemia and tissue hypoxia increase intestinal iron absorption and mitochondrial impairment in thalassemic patients. Regular blood transfusions improve hemoglobin levels but determine an iron overload that induces reactive oxygen species (ROS) overproduction. The aim of this study was to assess cellular oxidative damage by detection of ROS, lipid peroxidation, 8-oxo-dG, and mitochondrial transmembrane potential (Δψ(m)) in transfused and untransfused thalassemic patients. We have also evaluated genotoxicity by CBMN and comet assay. Our data show that ROS and lipid hydroperoxides are significantly higher in thalassemic patients than in controls, especially in untransfused thalassemia intermedia patients. Moreover, the latter have a significant decrease in Δψ(m) that highlights the energetic failure in hypoxic state and the ROS overproduction in the respiratory chain. 8-OHdG levels are higher in thalassemics than in controls, but do not differ significantly between the two patient groups. Both genotoxicity biomarkers highlight the mutagenic potential of hydroxyl radicals released by iron in the Fenton reaction. Values for percentage of DNA in the comet tail and micronuclei frequency, significantly higher in transfused patients, could also be due to active hepatitis C virus infection and to the many drug treatments. Our biomonitoring study confirms the oxidative damage in patients with thalassemia major and shows an unexpected cellular oxidative damage in untransfused thalassemic patients. In addition to iron overload, the results highlight the important role played by hypoxia-driven mitochondrial ROS overproduction in determining oxidative damage in ß-thalassemias.

Feasibility, reproducibility, and reliability for the T*2 iron evaluation at 3 T in comparison with 1.5 T.

This study aimed to determine the feasibility, reproducibility, and reliability of the multiecho T*(2) Magnetic resonance imaging technique at 3 T for myocardial and liver iron burden quantification and the relationship between T*(2) values at 3 and 1.5 T. Thirty-eight transfusion-dependent patients and 20 healthy subjects were studied. Cardiac segmental and global T*(2) values were calculated after developing a correction map to compensate the artifactual T*(2) variations. The hepatic T*(2) value was determined over a region of interest. The intraoperator and interoperator reproducibility for T*(2) measurements at 3 T was good. A linear relationship was found between patients' R *2 (1000/T*(2) ) values at 3 and 1.5 T. Segmental correction factors were significantly higher at 3 T. A conversion formula returning T*(2) values at 1.5 T from values at 3 T was proposed. A good diagnostic reliability for T*(2) assessment at 3 T was demonstrated. Lower limits of normal for 3 T T*(2) values were 23.3 ms, 21.1 ms, and 11.7 ms, for the global heart, mid-ventricular septum, and liver, respectively. In conclusion, T*(2) quantification of iron burden in the mid-ventricular septum, global heart, and no heavy-moderate livers resulted to be feasible, reproducible, and reliable at 3 T. Segmental heart T*(2) analysis at 3 T may be challenging due to significantly higher susceptibility artifacts.

Identification of alpha-thalassemia mutations in subjects from Eastern Sicily (Italy) with abnormal hematological indices and normal Hb A2.

We analyzed the prevalence of alpha-thalassemia mutations in 298 subjects from Eastern Sicily (Italy) with reduced mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH), normal HbA2 and HbF, and normal serum iron. In 131 subjects (43.9%) we found six different genotypes of alpha-thalassemia: -alpha3.7/alphaalpha (36.6%), -alpha3.7/-alpha3.7 (27.5%), -(MED)/alphaalpha (10.0%), -alpha20.5/alphaalpha (9.1%), alphaHphIalpha/alphaalpha (8.4%), alphaHphIalpha/alphaHphIalpha (6.1%), and -alpha3.7/alphaHphIalpha (2.3%). Our data underline that in Eastern Sicily populations, the molecular screening of alpha-thalassemia mutations and/or deletions may be useful to better characterize the clinically asymptomatic subjects with a slightly reduced MCV and MCH and normal iron status.

Increased protein carbonyl groups in the serum of patients affected by thalassemia major.

High oxidative stress status is known to be one of the most important factors determining cell injury in thalassemic patients and causing other serious medical complications, including a continuous proinflammatory status. The quantification of protein carbonyl groups in peripheral blood is widely used to measure the extent of oxidative modification. Thus, we measured serum concentrations of protein carbonyl groups in 30 patients affected by thalassemia major and in 15 healthy subjects. Strongly higher levels of protein carbonyl groups were measured in the blood from thalassemic patients than in that from healthy controls. Our findings evidence that thalassemic patients suffer from protein oxidative stress; the possibility of a role for carbonyl stress in the progression and severity of the disease needs further investigation.