Thirty-year experience in preventing haemoglobinopathies in Greece: achievements and potentials for optimisation.

OBJECTIVES: Beta thalassaemia major (ß-TM) and sickle-cell disease (SCD) are severe haemogobinopathies requiring life-lasting, advanced medical management. In the Mediterranean region, both conditions occur with high frequency. We assessed the efficacy of the National Program for the Prevention of Haemoglobinopathies in Greece during the last 30 yrs. METHODS: Data of affected births between 01/01/1980 and 31/12/2009 were collected in a nationwide scale, and expected vs. observed rates of new births were calculated and compared. In a subpopulation of affected births of Greek origin, the causes for occurrence of the new affected birth were also collected and analysed. RESULTS: Overall, the reduction in new cases was 81.1% and 84.6% for ß-TM and SCD, respectively. For ß-TM, a constant declining trend was recorded over the 30-yr period, whereas for SCD, a transient reversal was observed in the mid-1990s probably due to the significant influx of immigrants of African origin. Programme failure was 2.2 times more common among new ß-TM births of Greek origin compared to new SCD cases (P < 0.001). Unawareness and parental choice were more frequent in SCD compared to ß-TM (unawareness: OR = 1.4, P = 0.05, parental choice: OR = 1.9, P = 0.01). The main cause for programme failure was carrier misidentification and incorrect genetic advice for ß-TM and SCD, respectively. CONCLUSIONS: The ß-TM and SCD prevention programme in Greece has significantly reduced the numbers of new affected births. The outcomes could be optimised in groups of non-Greek origin, in carrier identification and by offering specialised genetic counselling.

Fok-I polymorphism of vitamin D receptor gene and the presence of renal dysfunction in patients with ß-thalassemia major.

Recent evidence supports the presence of renal dysfunction even among young patients with ß-thalassemia major. However, the possible genetic contribution has never been investigated. The aim of this study was to correlate the presence of Fok-I polymorphism of the vitamin D receptor gene with abnormal levels of early markers of renal impairment in children and young adults with thalassemia. Thirty-four patients (19 male and 15 female) with ß-thalassemia major on conventional treatment, with a mean decimal age of 14.62 ± 5.47 years (range: 5-22 years), were included in the study. Markers of renal function were determined in serum and in urine and patients were genotyped for Fok-I gene polymorphism. Genotype frequencies were similar to those previously reported for other populations: 47.06% of the patients were homozygous for the F allele, 41.18% were heterozygous, and 11.76% were homozygous for the f allele. A considerable number of patients demonstrated impaired renal function with increased serum cystatin C levels (29.41%), glomerular dysfunction with proteinuria (68%), as well as significant tubulopathy with hypercalciuria (73.08%), and increased levels of urinary ß(2)-microglobulin (29.41%). When patients were stratified according to Fok-I polymorphism, a significantly higher prevalence of abnormally increased serum levels of cystatin C was observed in patients being homozygous for the f allele (75%) compared with those being heterozygous (Ff) or homozygous for the F allele (14.29% and 31.25%, respectively, P = .02). Further studies are needed to confirm these preliminary results and elucidate the possible mechanisms involved.

Standard-dose intravenous anti-D immunoglobulin versus intravenous immunoglobulin in the treatment of newly diagnosed childhood primary immune thrombocytopenia.

BACKGROUND: We conducted a study to evaluate the efficacy of intravenous (IV) anti-D against IV immunoglobulin (IVIG) in newly diagnosed immune thrombocytopenia (ITP) in children and to identify the clinical characteristics of the children most likely to benefit from one or the other treatment. PROCEDURE: Children (6 mo to 14 y) with newly diagnosed ITP and a platelet count <20,000/µL were treated either with a single bolus dose of 50 µg/kg IV anti-D or with 0.8 to 1 g/kg IVIG in a randomized manner. RESULTS: Twenty-five patients, mean age of 6.8 years, were treated either with IV anti-D (n=10) or with IVIG (n=15). Both drugs were equally efficient in raising the platelet count above 20,000/µL at 24 hours posttreatment. Children who presented with bleeding stage 1 or 2 (no mucosal bleeding) responded better to IVIG treatment, in terms of an increase in platelet count at 24 hours posttreatment (P=0.04). Hemoglobin drop was greater in the anti-D group (P=0.002). CONCLUSIONS: A single bolus dose of 50 µg/kg of IV anti-D is a safe and effective first-line treatment in newly diagnosed ITP in childhood and mucosal bleeding is a poor prognostic factor for treatment with IVIG.

Fok-I gene polymorphism of vitamin D receptor in patients with beta-thalassemia major and its effect on vitamin D status.

Most of the biological actions of vitamin D are mediated by an intracellular receptor (VDR) in which several single nucleotide gene polymorphisms have been identified. Vitamin D deficiency is increasingly identified among thalassemic patients and recent evidence links it with myocardial iron accumulation. The aim of this work was to assess the distribution of the Fok-I polymorphism of the VDR gene among Greek children and young adults with beta-thalassemia major and to investigate its association with 25(OH)D(3) and 1,25(OH)(2)D(3) serum levels. Sixty-nine thalassemic patients (35 females and 34 males), with a mean age of 23·05±6·07 years, participated in the study. Genotype frequencies of Fok-I were similar to those previously reported for other populations; 44·9% of the patients were homozygotes for F allele, 43·5% were heterozygotes and 11·6% were homozygotes for the f allele. Low levels of serum 25(OH)D(3) were recorded, as 41 patients (59·4%) were below the cut-off limit of 50 nmol/l that determines deficiency, whereas, levels of 1,25(OH)(2)D(3) showed wide variability ranging from deficiency (≤50 pmol/l) in 34 patients (49·3%) to excess (≥125 pmol/l) in 13 patients (18·8%). When stratifying patients according to serum 1,25(OH)(2) D(3) concentrations, a higher prevalence of the f allele was observed in the deficiency group (P = 0·03). A comparison of the serum concentrations of the two vitamin D metabolites produced a trend towards a negative correlation (r = -0·204, P = 0·09). Further studies are required to assess the genetic contribution to the regulation of vitamin D metabolites in the serum of patients with beta-thalassemia major.

Renal dysfunction in patients with beta-thalassemia major receiving iron chelation therapy either with deferoxamine and deferiprone or with deferasirox.

There are limited studies on renal involvement in beta-thalassemia, mainly involving patients on deferoxamine, reporting both glomerular and tubular dysfunction. The aim of the present study was to investigate renal involvement in young thalassemia patients, using both conventional and early markers of renal dysfunction, and to correlate findings to iron chelation therapy. Forty-two patients aged 4-23 years were studied and, for analysis purposes, were divided into two groups based on chelation therapy (group A receiving deferasirox and group B receiving deferoxamine and deferiprone combination therapy). In addition to conventional renal biochemistries, creatinine clearance, estimated glomerular filtration rate, serum cystatin C (Cys C), fractional excretion of sodium, tubular phosphorus reabsorption and urine calcium, protein, beta(2)-microglobulin (beta(2)-MG) and glucose levels were measured. A considerable number of patients demonstrated impaired renal function with elevated Cys C levels (36%), glomerular dysfunction with proteinuria (24%) and tubulopathy with hypercalciuria (35.5%) and elevated excretion of beta(2)-MG (33.5%). Renal involvement seems to be present even in young patients with beta-thalassemia, therefore, routine use of early markers of renal dysfunction is recommended. Further studies are needed in order to investigate the role of new chelators in tubular function parameters.

Elevated serum parathormone levels are associated with myocardial iron overload in patients with beta-thalassaemia major.

OBJECTIVES: Despite advances in conventional treatment, iron-induced cardiomyopathy is still the most frequent cause of death among patients with beta-thalassaemia major. Recent studies have correlated increased myocardial iron content to decreased levels of vitamin D in thalassaemic patients. The aim of this study was to measure parathormone (PTH) and metabolites of vitamin D and consequently to investigate whether these parameters predispose to myocardial iron overload in patients with beta-thalassaemia major. METHODS: In 62 patients (29 M and 33 F, mean age: 22.79 +/- 6.18 yr) with beta-thalassaemia major levels of intact parathormone (iPTH) and vitamin D metabolites [25(OmicronH)D(3) and 1,25(OmicronH)(2)D(3)] were measured in serum. Additionally, estimation of myocardial iron content was performed by magnetic resonance imaging, whereas mean serum ferritin concentrations were calculated for 1 yr prior to the study. RESULTS: Results showed markedly decreased levels of serum 25(OH)D(3) in 37 patients (60%), whereas 7 patients (11%) had borderline 25(OH)D(3) levels (between 50 and 75 nmol/L). Serum iPTH levels were significantly higher in patients having increased myocardial iron compared to those having normal myocardial iron (44.04 +/- 22.09 pg/mL vs. 31.39 +/- 14.30 pg/mL, P = 0.017). Multivariant regression analysis identified PTH levels as the major predictor of increased myocardial iron.

MRI assessment of liver iron content in thalassamic patients with three different protocols: comparisons and correlations.

Our aim was to assess liver iron content, in thalassaemic patients, by using three different MR protocols and compare their data. Ninety-four thalassaemic patients (44 M and 50 F, mean age 25.82 +/- 8.3 yrs), were enrolled in the study. In each patient, three measurements of the liver iron content were performed, with the use of a single imager, equipped with a 1.5 Tesla magnet. Liver R2* was measured on gradient-echo sequence. Calculation of MR-HIC values was based on an algorithm using liver to muscle (L/M) ratios in five axial gradient-echo sequences. Finally, determination of liver R2 employed a 16-echo, spin-echo pulse sequence. Additionally, myocardial R2* value was determined for each patient. Results showed that all three magnetic resonance imaging (MRI) methods were highly correlated to each other and significantly correlated to serum ferritin concentrations. Liver R2 method showed an increased sensitivity in detecting liver iron contents in the upper range. No correlation occurred between each liver MRI parameter and myocardial R2* values. Finally, we managed to provide formulae for equating values obtaining with any of these three MRI methods.

Poor correlations between measurements of bone quality by quantitative ultrasound sonography and dual energy X-ray absorptiometry in patients with beta-thalassaemia major.

OBJECTIVES: Osteopenia/osteoporosis is a major component of morbidity even in young patients with beta-thalassaemia major. Dual energy X-ray absorptiometry (DXA) is the reference method for determining bone mineral density (BMD). Quantitative ultrasound sonography (QUS) for bone measurement is a relatively new, inexpensive and radiation-free method that could serve as an alternative to DXA. Our aim was to assess bone status in thalassaemic patients both with QUS and DXA and, consequently, to investigate the degree of correlation between the two methods. METHODS: Thirty-three patients (15 male and 18 female) with beta-thalassaemia major, regularly transfused and systematically iron-chelated, participated in the study. Mean age was 22.0 +/- 8.0 yr (range: 6.5-41.0 yr). All patients were evaluated with QUS at radius and tibia and had DXA scan at lumbar spine vertebrae (L2-L4), whereas 20 patients were additionally assessed with DXA at the left hip (femoral neck, trochanter region and Ward's triangle). RESULTS: Results were expressed as Z-scores compared with sex- and age-matched population. Lowest mean Z-scores measured with DXA were recorded at lumbar spine and Ward's triangle (-1.1 +/- 1.13 and -0.95 +/- 1.07, respectively). Lowest mean QUS-derived Z-scores were measured at radius, statistically significant compared with Z-scores measured at tibia (-0.6 +/- 1.1 vs. 0.4 +/- 1.1, P < 0.001). QUS measurements at radius were significantly correlated to QUS measurements at tibia (r = 0.51, P = 0.002). The latter were correlated to BMD measured at lumbar spine (r = 0.516, P = 0.002) and at trochanter region (r = 0.646, P = 0.003). All BMD measurements at hip were significantly correlated to each other. Lumbar spine BMD was correlated to BMD at femoral neck (r = 0.607, P = 0.003) and to BMD at Ward's triangle (r = 0.438, P = 0.027). Finally, no agreement was recorded between the two methods in identifying thalassaemic patients at risk for osteoporosis (kappa = 0.203, P = 0.04). CONCLUSION: Quantitative ultrasound sonography could not serve as an alternate to DXA.

Deferiprone-related arthropathy of the knee in a thalassemic patient: report of a case and review of the literature.

Deferiprone (DFP), the first oral iron chelator, has been used in patients with beta-thalassemia major to reduce serum ferritin levels and total iron burden, leading to decreased cardiac iron levels. Major side effects include embryotoxicity, agranulocytosis, zinc deficiency and gastrointestinal disorders, while arthropathy is rarely reported. Herein, we present a 29-year-old male patient with beta-thalassemia major, who developed severe arthritis of both knees while under deferiprone therapy. Arthritis was managed successfully with non-steroid antiinflammatory drugs after DFP withdrawal.

Normal lumbar bone mineral density in optimally treated children and young adolescents with beta-thalassaemia major.

OBJECTIVE: Osteopenia/osteoporosis of multi-factorial pathogenetic mechanism is reported to be a significant cause of morbidity in adult patients with beta-thalassaemia major. Even in young patients, decreased Bone Mineral Density (BMD) values are a consistent finding in the literature. This study was performed in order to assess BMD in children and young adults with beta-thalassaemia major, regularly transfused and sufficiently chelated, along with auxological, clinical and laboratory parameters. DESIGN: Thirty-five young thalassaemic patients (19 F, 16 M, aged 5-20 yr) were studied. Lumbar BMD was assessed by dual X-ray absorptiometry (DXA) and Z-scores were calculated according to bone density values using age- and sex-matched normal population. None of the patients presented with clinical or laboratory signs of endocrinopathy and none was receiving hormonal replacement therapy. RESULTS: All BMD Z-scores were within normal range, with a mean Z-score of 0.42 for girls and -0.41 for boys (statistically significant gender difference, p=0.018). When correlated with age, a decline in Z-scores was observed, indicating a delay in bone mass acquisition with advancing age in the thalassaemic group compared to controls. CONCLUSIONS: Optimal conventional treatment prevents the manifestation of osteopenia/osteoporosis during the first two decades of life in patients with beta-thalassaemia major. However, close surveillance with regular screening, preventive intervention and early management of possible endocrine complications are essential in order to secure normal bone health during adulthood and improve quality of life in the thalassaemic population.

Tibial pseudotumor in a child with hemophilia.

Hemophilic pseudotumor is an uncommon complication seen in approximately 1-2% of patients with severe hemophilia. Hemophilic pseudotumors are distinguished into two subdivisions based on location, proximal or distal. Plain x-rays and CT are useful in diagnosis, but MR imaging is the diagnostic test of choice because of its sensitivity to the various blood products. The choice of therapy depends on many parameters, such as the size of the tumor, the age of the patient, and the relation with underlying organs. In most cases of asymptomatic hemophilic pseudotumor, conservative treatment with administration of missing factor as well as immobilization is recommended. The authors describe a 13-year-old boy with severe hemophilia A, who presented with a tibial pseudotumor a few months after an injury. He was conservatively treated for a long period, with daily administration of recombinant factor VIII. His clinical condition improved shortly after therapy induction, but radiological improvement has been moderate. Case history, imaging findings, and therapeutic options are discussed.

Urinary iron excretion in young thalassemic patients receiving combined chelation treatment with deferoxamine and deferiprone.

To assess and compare the individual effect of different chelation agents on urinary iron excretion (UIE), we asked every patient, receiving combined chelation treatment with deferiprone (DFP) and deferoxamine (DFO), to provide four 24-hours urine samples; 2 samples were collected during days when patient was receiving only DFP, whereas the other 2 were collected when both chelation agents were administrated. Thirty young patients (15 males and 15 females) with beta-thalassemia major and a mean age of 18.54+/-4.62 years participated in the study. Mean serum ferritin concentrations were calculated 1 year prior and 1 year after the urine collection. A significant reduction in ferritin (P=0.001) was shown in the whole patients' series. Combined administration of DFO and DFP resulted in a statistically significant higher UIE than DFP alone (P=0.0007). On an individual basis, DFO and DFP resulted in a median 2.3-fold increase in UIE compared to monotherapy with DFP, ranging from 0.28 to 7.34-fold. Despite this wide variability, combined chelation treatment with DFO and DFP seems to act additively in the majority of the patients, whereas in some patients the huge increase in UIE with DFO and DFP can only be attributed to a synergistic effect.

Evaluation of serum tumour markers concentrations in patients with homozygous beta-thalassaemia in relation to demographical, clinical and biochemical parameters.

Increased life expectancy in patients with homozygous beta-thalassaemia consequently increases the risk for neoplastic diseases. This study was conducted to assess the levels of five common tumour markers in thalassaemic patients and to investigate possible correlations to demographical, clinical and laboratory data. Eighty-five patients (44 female and 41 male) with homozygous beta-thalassaemia (mean age = 27.92 +/- 12.5), on regular blood transfusions and adequate chelation treatment, and 60 sex and age- matched healthy controls were enrolled in the study. Blood samples for the determination of carcinoma antigen (CA) 15.3, CA 125, CA 19.9, carcinoembryonic antigen (CEA) and alpha-fetoprotein (a-FP) were collected from every subject. Results showed that 69% of the thalassaemic patients had abnormal levels of CA 15.3, whereas only sporadic cases had increased levels of CA 125 and CA 19.9. On the contrary, all controls had normal levels of CA 15.3, CA 19.9 and CA 125. CEA and a-FP were within reference ranges both in the thalassaemic and in the control group. Levels of CA 15.3 were significantly lower in patients aged less than 20 years compared to older patients. Male patients had significantly increased levels of CA 15.3 compared to female patients. Relatively recent studies show an increased expression of CA 15.3 on progenitor cells of the erythroid lineage and increased amounts of circulating progenitor cells even in well-transfused thalassaemic patients. However, it seems that there are also other factors contributing to this phenomenon. In conclusion, our results indicate that CA 15.3 seems to be an unreliable marker of occult malignancy in patients with beta-thalassaemia. However, more studies are needed to support these preliminary results.

Evolution of OGTT in patients with beta-thalassaemia major in relation to chelation therapy.

Glucose metabolism disturbances are frequently reported among patients with beta-thalassaemia major on conventional treatment consisted of regular blood transfusions and adequate chelation treatment. Aim of this study was to evaluate the evolution of oral glucose tolerance test (OGTT) in thalassaemic patients in relation to their chelation treatment. Data from two OGTTs performed with an interval of 2 years were studied retrospectively. Patients considered eligible for this study were those who maintained unchanged chelation treatment and did not receive any anti-diabetic agent during the last 2 years. Thirty-one patients (16 M and 15 F) were enrolled with a mean age of 23.73+/-4.23 years at the end of the study. Patients were divided into three groups concerning chelation treatment. First group was receiving deferoxamine (DFO) by an 8-hourly subcutaneous infusion five-six times a week, second group was chelated with deferiprone (DFP) at a daily dose of 75 mg/kg orally and the third group was receiving combined therapy with DFO (3 days/week) and DFP (daily). At the time of the first OGTT, 26 patients (84%) were found to have normal OGTT; three of them showed an impaired glucose tolerance during second test (one was chelated with DFP and two were receiving combined therapy). None of the five patients with impaired glucose metabolism during the first test became diabetic. On contrary, one patient receiving combined therapy managed to normalize his second OGTT. In contrast with the overall trend of a deteriorating glucose tolerance in the whole patient series, the group receiving combined therapy managed to increased beta-cell function index and decreased insulin resistance index, although not statistically significant when compared to other groups. Further studies are needed to support these preliminary results.

MRI for the determination of pituitary iron overload in children and young adults with beta-thalassaemia major.

Hypogonadism, resulting from iron-induced pituitary dysfunction, is the most frequently reported complication in patients with beta-thalassaemia major. The aim of this study was to evaluate pituitary Magnetic Resonance Imaging (MRI) signal intensity reduction, on T2*-weighted images, as a marker of pituitary iron overload. Thirty patients (13 females and 17 males, mean age: 16.6+/-4.1) with beta-thalassaemia major on conventional treatment and 13 healthy volunteers (7 females and 6 males, mean age: 11+/-4.51 years) were studied with T2*-weighted images of the anterior pituitary using a 1.5T unit. Four thalassaemic patients (2 females and 2 males) had clinical hypogonadism and required hormonal replacement treatment. Results revealed a statistically significant reduction of pituitary signal intensity in the thalassaemia group compared to controls (p<0.001). Moreover, hypogonadal patients had significantly decreased MRI values compared to thalassaemic patients without hypogonadism (p=0.017). Relatively decreased adeno-hypophyseal MRI signal intensity was recorded in pubertal thalassaemic patients. A significant negative correlation was observed between pituitary MRI values and age (r=-0.67, r(2)=0.443, p=0.001), whereas ferritin levels and pituitary MRI values were moderately correlated (r=-0.56, r(2)=0.32, p=0.08) in adult thalassaemic patients. In conclusion, pituitary MRI indices as measured on T2*-weighted images seem to reflect pituitary iron overload and could, therefore, be used for a preclinical detection of patients who are in greater danger of developing hypogonadism.

Four-year evaluation of myocardial and liver iron assessed prospectively with serial MRI scans in young patients with beta-thalassaemia major: comparison between different chelation regimens.

This study was conducted in order to assess myocardial and liver iron concentrations (LICs) using serial magnetic resonance imaging (MRI) scans in patients with beta-thalassaemia major, over a 4-yr period, and consequently to compare the effectiveness of different chelation regimens. Fifty children and young adults with beta-thalassaemia major (27 boys and 23 girls) were recruited (mean age: 14.74 +/- 3.67 yr). All patients underwent detailed clinical examination, electrocardiography, echocardiography, myocardial and liver MRI at the beginning of the study, 2 and 4 yr after. Additionally, serum ferritin levels were regularly measured and data regarding LICs assessed by percutaneous liver biopsy were available in 26 patients. Both myocardial and liver MRI values showed a moderate inverse correlation with age (r = -0.379, P < 0.001 and r = -0.376, P < 0.001, respectively). Liver MRI was better correlated with serum ferritin concentrations (r = -0.342, P < 0.001) than myocardial MRI (r = -0.186, P = 0.011). Liver MRI values were highly correlated with LICs derived from percutaneous liver biopsy (r = -0.863, P < 0.001), whereas myocardial MRI values did not correlate at all with measurements derived from echocardiography. Regarding iron chelation treatment, patients receiving combined therapy with deferiprone and deferoxamine (DFO) significantly reduced myocardial iron overload during the 4-yr study period, whilst patients in monotherapy with DFO showed a significant increase in LIC.

comparison of effects of different long-term iron-chelation regimens on myocardial and hepatic iron concentrations assessed with T2* magnetic resonance imaging in patients with beta-thalassemia major.

The aim of this study was to compare the effect of different long-term chelation regimens on heart and liver iron stores with the use of T2* magnetic resonance imaging (MRI) in patients with transfusion-dependent beta-thalassemia major. Sixty-four patients (28 men, 36 women; mean age, 26.49 +/- 5.8 years) were enrolled in the study. The 3 groups were based on the chelation therapy received. The first group (19 patients) received deferiprone (DFP) (75 mg/kg per day orally), the second group (23 patients) received deferoxamine (DFO) (30-50 mg/kg per day subcutaneously at least 5 times/week), and the third group (22 patients) received a combination of DFO (30-50 mg/kg per day, 2-3 days/week) and DFP (75 mg/kg per day, 7 days/week). MRI scans were acquired with an imager equipped with a 1.5 T magnet, and the data included myocardial and hepatic iron measurements obtained by means of T2*, and ventricular volumes and ejection fractions obtained with standard cardiovascular MRI techniques. The results revealed that the DFP and the combined groups had significantly less myocardial iron than the DFO group (mean myocardial T2*, 35.77 +/- 18.3 milliseconds and 38.05 +/- 15.3 milliseconds versus 23.77 +/- 13 milliseconds [P = .02, and P = .001], respectively). On the contrary, the DFP group had a significantly higher hepatic iron content than the DFO and combined groups (mean hepatic T2*, 3.29 +/- 2.5 milliseconds versus 8.16 +/- 8.4 milliseconds and 11.3 +/- 10.9 milliseconds [P = .014, and P = .003], respectively). No correlation was observed between myocardial T2* and hepatic T2* values (r = -0.043; P = .37). Myocardial T2* values were inversely correlated with age (r = -0.249; P = .024) and positively correlated with both left and right ventricular ejection fractions (r = 0.33 [P = .004], and r = 0.279 [P = .014], respectively). Finally, liver T2* was strongly and inversely correlated with serum ferritin concentration (r = -0.465; P = .001). In conclusion, combined chelation therapy seems to sum the beneficial effects of DFO and DFP with respect to hepatic and myocardial iron. Because myocardial iron is not related to measurements of serum ferritin or hepatic T2*, important decisions on clinical management relating to cardiac risk should not rely on these conventional parameters. Thus, the use of MRI for assessing myocardial iron should be adopted in the routine clinical management of patients with beta-thalassemia major.