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We report two very rare changes in the second intron of the HBB gene, a substitution at nucleotide (nt) 726 [IVS-II-726 (A>G) (β+), NM_000518, HBB: c.316-125A>G] and a deletion of a cytosine at nt 809 [IVS-II-809 (-C) (β), NM_000518, HBB: c.316-42delC] identified during the screening program for hemoglobinopathies in the resident Sicilian population. The purpose of this study was to evaluate the clinical implication of these rare changes, particularly in coinheritance with known mutations in the globin clusters, in order to conduct an appropriate genetic counseling for at-risk couples. Molecular analysis detected the first rare nt substitution in two cases in simple heterozygosity and in two cases in association with other known mutations on globin genes, while the deletion was identified in a pregnant woman, carrier of β-thal, and in her fetus at prenatal diagnosis (PND) for hemoglobinopathies. The present study emphasizes the importance of sharing the observed changes in the globin gene cluster, especially in the case of new or rare undefined mutations, in order to facilitate the determination of their phenotypic expression and possible interactions with known molecular defects.

BackgroundThalassaemia and variant haemoglobin are the most common severe monogenic disorders worldwide.AimsTo develop prenatal diagnosis programmes for the prevention of the most important haemoglobin disorders and identify healthy carriers of thalassaemia.MethodsSequencing analysis was used to obtain complete data on gene structure and to correlate specific phenotypic expression with mutations, especially for new or very rare mutations in globin genes.ResultsA rare single nucleotide variation, HBB:c.93–23T>C, located in nucleotide 108 of the first intervening sequence of the HBB gene, was identified. This variation was previously reported but its clinical significance was not known. Six heterozygous patients had this nucleotide variation and eight further cases co-inherited it together with other defects in the globin genes. Heterozygous subjects for this substitution showed normal haematological and electrophoretic features, whereas subjects who were compound heterozygotes for this mutation and another defect in globin genes showed the classic phenotype of a healthy carrier.ConclusionThis nucleotide can be considered a single nucleotide polymorphism and not a thalassaemic mutation that reduces the production of haemoglobin. This is another example of a very rare nucleotide variation. Knowledge of this is important so that appropriate genetic counselling can be carried out of a couple potentially at risk, where one of the partners is a carrier of β-thalassaemia and the other is carrier of a nucleotide variation.

Objective Celocentesis, which involves aspiration of celomic fluid at 7–9 weeks' gestation, can potentially provide early prenatal diagnosis of single-gene disorders. The main barrier to wide acceptability of this technique is contamination of the sample by maternal cells. This problem can be overcome through selection of embryo-fetal erythroid precursors, which are found in celomatic fluid. Method Embryo-fetal erythroid precursors were selected by an anti-CD71 MicroBeads method or by direct micromanipulator pickup of the cells selected on the basis of their morphology. Results In our series of 302 singleton pregnancies at high risk for hemoglobinopathies, Celocentesis provided a sample of celomic fluid in all cases. In 100 (33.1%) samples, maternal contamination was absent or very low ( 60%, and selection of embryo-fetal cells was achieved by micromanipulation. In all 302 cases, there was concordance between DNA obtained from celomic fluid samples and fetal or newborn DNA. Conclusions Celocentesis can be a reliable procedure for earlier prenatal diagnosis of fetal monogenic diseases. © 2016 John Wiley & Sons, Ltd.

Hereditary aceruloplasminemia is a rare disease characterized by iron overload and neurodegeneration. Aceruloplasminemia is due to the absence/deficiency of ceruloplasmin, responsible for iron overload in liver, pancreas and other organs. We report the case of an Italian patient with hyperferritinemia, diabetes and hepatic iron excess, suspected to be affected by aceruloplasminemia. Patient underwent brain magnetic resonance imaging with and without paramagnetic medium contrast, which showed a hypointesity due to iron storage. The presence of a neurological disease and iron storage in the brain has led to assume the suspect of the aceruloplasminemia disease. We confirmed this hypothesis by the identification of a new nonsense mutation in exon 12 ceruloplasmin gene at codon 748, in homozygous status. When the diagnosis of ACP was established, the patient started chelation therapy with 75 mg/Kg/day deferiprone (DFP). Two months of starting therapy with DFP, serum ferritin values decreased to 693 ng/ml; the patient well tolerated the drug, and there have been no adverse events. Although rare, aceruloplasminemia should be considered in the differential diagnosis of unexplained iron overload.

In sickle cell disease (SCD), hydroxyurea (HU) treatment decreases the number of vaso-occlusive crisis (VOC) and acute chest syndrome (ACS) by increasing fetal hemoglobin (HbF). Data are lacking regarding the frequency of HU dose modification or whether sub-therapeutic doses (

Background: Thalassemia minor (Tm) individuals, are generally considered healthy. However, the prognosis of Tm individuals has not been extensively studied. The aim of this study was to evaluate the prognosis of Tm versus controls without β-thalassemia carrier state. Methods: A total of 26,006 individuals seeking thalassemia screening at the AOOR Villa Sofia-V. Cervello, Palermo (Italy) were retrospectively studied. Logistic penalised regression model was used to estimate risk of potential complications and survival techniques were used to study mortality. Results: We identified a total of 4943 Tm and 21,063 controls. Tm was associated with significantly higher risks of hospitalisation for cirrhosis (OR 1·94, 95% CI 1·30 to 2·90, p = 0·001), kidney disorders (OR 2·11, 95% CI 1·27 to 3·51, p = 0·004), cholelithiatis (OR 1·39, 95% CI 1·08 to 1·79, p = 0·010), and mood disorders (OR 2·08, 95% CI 1·15 to 3·75, p = 0·015). No statistically difference in life expectancy between thalassemia minor and control group was found (HR 1·090, 95% CI 0·777 to 1·555, p < 0·590; log-rank test p =.426). Conclusion: This study shows that Tm affects the prognosis of Tm carriers regarding health expectation. Probably, iron overload and anaemia for several years may be at the basis of these effects.

Blood transfusion and iron chelation currently represent a supportive therapy to manage anemia, vasculopathy and vaso-occlusion crises in Sickle-Cell-Disease. Here we describe the first 5-year long-term randomized clinical trial comparing Deferiprone versus Deferoxamine in patients with Sickle-Cell-Disease. The results of this study show that Deferiprone has the same effectiveness as Deferoxamine in decreasing body iron burden, measured as repeated measurements of serum ferritin concentrations on the same patient over 5-years and analyzed according to the linear mixed-effects model (LMM) (p=0.822). Both chelators are able to decrease, significantly, serum ferritin concentrations, during 5-years, without any effect on safety (p=0.005). Moreover, although the basal serum ferritin levels were higher in transfused compared with non-transfused group (p=0.031), the changes over time in serum ferritin levels were not statistically significantly different between transfused and non-transfused cohort of patients (p=0.389). Kaplan-Meier curve, during 5-years of study, suggests that Deferiprone does not alter survival in comparison with Deferoxamine (p=0.38). In conclusion, long-term iron chelation therapy with Deferiprone was associated with efficacy and safety similar to that of Deferoxamine. Therefore, in patients with Sickle-Cell-Disease, Deferiprone may represent an effective long-term treatment option.

The aim of this study is to update the incidence and the distribution of the globin gene defects causing β-thalassemia and abnormal hemoglobins in Sicily. The data derived from a total of 8875 beta-thalassemia alleles and 1330 variant hemoglobin chromosomes studied in Sicily from 1990 during a hemoglobinopathy control program. Fifty-four beta-globin gene defects were characterized, involving 30 different beta-thalassemia mutations and 24 variant hemoglobins. Eight of 30 β-thalassemia defects accounted for 95.11% of examined alleles while other beta-globin gene defects were found at lower frequencies (

At 5-12 weeks of gestation the amniotic sac is surrounded by celomic fluid which contains cells of fetal origin. This fluid can be sampled by celocentesis, which involves the ultrasound-guided insertion of a needle through the vagina. The aim of this study was to examine the feasibility of prenatal diagnosis of hemoglobinopathies from the celomic fluid using a specific protocol. Celocentesis was performed at 7-9 weeks' gestation in 26 singleton pregnancies at risk for hemoglobinopathies. In 25 cases more than 30 fetal cells were recovered from the celomic fluid and in all these cases molecular analysis for hemoglobinopathies was possible and the results were confirmed by subsequent chorionic villus sampling or amniocentesis. The results of this study suggest that reliable diagnosis of thalassemia syndromes can be performed from 7 weeks' gestation by celocentesis. Further work is necessary to demonstrate the safety of celocentesis, although further work is necessary to improve widespread use and safety of the procedure..

Hydroxycarbamide (HC) is a pharmacological agent capable of stimulating fetal haemoglobin (HbF) production during adult life. High levels of HbF may ameliorate the clinical course of β-thalassaemia and sickle cell disease. The efficacy of HC for the treatment of thalassaemia major and thalassaemia intermedia is variable. Although an increase of HbF has been observed in most patients, only some patients experience significant improvement in total haemoglobin levels. This study aimed to determine the effectiveness and safety of short- (1 year) and long-term (mean follow-up 68 months) HC treatment in 24 thalassaemia intermedia patients. Additionally, we evaluated if primary erythroid progenitor cells cultured from treated patients responded to HC treatment in a manner similar to that observed in vivo. Our results confirm a good response to HC after a short-term follow-up in 70% of thalassaemia intermedia patients and a reduction of clinical response in patients with a long follow-up. Erythroid cultures obtained from patients during treatment reproduced the observed in vivo response. Interestingly, haematopoietic stem cells from long-term treated patients showed reduced ability to develop into primary erythroid cultures some months before the reduction of the 'in vivo' response. The mechanism of this loss of response to HC remains to be determined.

Celocentesis, which involves aspiration of celomic fluid at 7-9 weeks' gestation, can potentially provide early prenatal diagnosis of single-gene disorders. The main barrier to wide acceptability of this technique is contamination of the sample by maternal cells. This problem can be overcome through selection of embryo-fetal erythroid precursors, which are found in celomatic fluid.Embryo-fetal erythroid precursors were selected by an anti-CD71 MicroBeads method or by direct micromanipulator pickup of the cells selected on the basis of their morphology.In our series of 302 singleton pregnancies at high risk for hemoglobinopathies, Celocentesis provided a sample of celomic fluid in all cases. In 100 (33.1%) samples, maternal contamination was absent or very low (5%), and unambiguous results were obtained without the need for any preliminary procedures. In 160 (53%) cases, the contamination was between 5% and 60%, and selection of embryo-fetal erythroid precursors was successfully achieved by anti-CD71 MicroBeads. In 42 (13.9%) cases, the contamination was60%, and selection of embryo-fetal cells was achieved by micromanipulation. In all 302 cases, there was concordance between DNA obtained from celomic fluid samples and fetal or newborn DNA.Celocentesis can be a reliable procedure for earlier prenatal diagnosis of fetal monogenic diseases.

Summary Introduction During an intensive screening program aimed at identifying the healthy carriers of thalassemia and the couples at risk of bearing an affected fetus, a rare single nucleotide variation (SNV), CAP + 1570 T > C (HBB:c*96T > C), located 12 nucleotides upstream of the polyadenylation signal in 3'UTR of the beta globin gene was identified. It was previously reported as a β+ thalassemia mutation and later as a plain polymorphism. Methods Genotype identification of globin gene mutations was carried out using sequencing analysis, GAP-PCR, and MLPA methods. Results CAP + 1570 T > C (HBB:c*96T > C) was found in 39 heterozygotes, in one case in homozygous state and in thirteen cases of co-inheritance of this nucleotide substitution with other mutations in globin genes. Carriers of this mutation showed a ‘silent’ phenotype without appreciable microcytosis and hypochromia, so they cannot be differentiated from noncarrier individuals. Compound heterozygotes for this mutation and severe β-thal mutations showed a variable phenotype ranging from β-thal carrier to mild form of β-thalassemia intermedia, revealing new aspects and allowing to better understand the clinical implications of this nucleotide substitution that can be classified as a silent β-thalassemic defect. Conclusion Data reported in this study indicate the need of investigating partner of β-thalassemia carrier by complete sequencing analysis of β-globin gene and of providing an appropriate genetic counseling for couples at risk undergoing prenatal diagnosis.

Hepatocellular carcinoma (HCC) frequently complicates hepatic cirrhosis secondary to viral infection or iron overload. Therefore, patients affected by thalassaemia syndromes have a theoretically high risk of developing the tumour. We collected data on patients attending Italian centres for the treatment of thalassaemia. Twenty-two cases of HCC were identified; 15 were male. At diagnosis, the mean age was 45 +/- 11 years and the mean serum ferritin was 1764 +/- 1448 microg/l. Eighty-six percent had been infected by hepatitis C virus. Nineteen of 22 cases were diagnosed after 1993, suggesting that this problem is becoming more frequent with the aging population of thalassaemia patients.

Summary Background Haemoglobinopathies are a major public health problem in Sicily: it was estimated a frequency of 1/245 couples are at risk of haemoglobinopathies. This paper reviews legislative actions, prevention activities, carrier screening, genetic counselling, foetal sampling and laboratory methodology analysis evolution reporting the results of 30 years of prevention actions to assess the efficiency of our preventative programme in the control of haemoglobinopathies in Sicily. Methods This programme consisted principally of five phases: legislative actions, public awareness campaign, carrier screening, genetic counselling and prenatal diagnosis. Results These programmes have been very effective, which we can see from a greater public awareness of thalassaemia and its prevention in the target population furthermore by a marked decline in the incidence of thalassaemia major and sickle cell anaemia from 1 in 245 live births in the absence of prevention to 1 in 2000, with a reduction in about 85%. The residual cases were because of a conscious choice by expecting parents in relation to improved life expectancy as well as improved quality of life of the affected patients. Conclusion The study suggests that public health authorities should act and invest in a similar programme for prevention of thalassaemia, as well as in relation to the increased survival of patients and the consequent organ complications.

In patients with β-thalassemia and sickle cell syndromes there is an important secondary iron overload due to regular blood transfusions and increased duodenal iron absorption. As in genetic hemochromatosis, also the secondary iron storage leads to tissue injury that involves all the major organs: liver, heart, kidney, endocrine glands. At present, in patients with β-thalassemia and sickle cell syndrome, iron chelation therapy is widely used for the treatment of secondary hemochromatosis, to limit the toxic effects of iron overload. In order to maintain the correct homeostasis, several genes are involved in the metabolic pathways of iron, including HFE, FPN (ferroportin) and TF (transferrin). In this study we analyzed the genes HFE, FPN and TF, to assess their possible effects on response to therapy with deferasirox and deferiprone, either as monotherapy or in combination therapy in a cohort of patients with β-thalassemia and sickle cell syndromes.

Prenatal diagnosis of hemoglobinopathies involves the study of fetal material from blood, amniocytes, trophoblast coelomatic cells and fetal DNA in maternal circulation. Its first application dates back to the 70s and it involves globin chain synthesis analysis on fetal blood. In the 1980s molecular analysis was introduced as well as amniocentesis and chorionic villi sampling under high-resolution ultrasound imaging. The application of direct sequencing and polymerase chain reactionbased methodologies improved the DNA analysis procedures and reduced the sampling age for invasive prenatal diagnosis from 18 to 16–11 weeks allowing fetal genotyping within the first trimester of pregnancy. In the last years, fetal material obtained at 7–8 weeks of gestation by coelocentesis and isolation of fetal cells has provided new platforms on which to develop diagnostic capabilities while non-invasive technologies using fetal DNA in maternal circulation are starting to develop.

Fifteen beta-thalassemia intermedia patients, not requiring chronic transfusional therapy, were monitored in order to check their antioxidant status, and the lipid oxidation products in plasma, LDL, and erythrocytes before and during a 9-month oral treatment with 600 mg/day vitamin E. The low level of vitamin E, and high level of malondialdehyde in plasma clearly tended to normalize after three months (P < .001), and were quite similar to control after six months. The abnormally low level of vitamin E in LDL and the four times higher than control basal level of conjugated dienes (LDL-CD), were not modified after three months of treatment. Significant changes of LDL-VE (P < .05) and of the basal LDL-CD (P < .001) were evident after six months. LDL-VE was within the normal range after nine months, whereas LDL-CD still appeared twice as higher than control. Plasma vitamin A, ascorbate, beta-carotene, and lycopene increased markedly at the end of the trial (P < .005). The level of vitamin E in red blood cells was normalized after six months of supplementation. A decrease of the baseline value of conjugated dienes was observed after nine months, although it remained 1.4-fold higher than control. The RBC count and hematocrit appeared higher at the end of the trial (P < .05 and P < .001, respectively). The hemoglobin value did not show variations. A shift to normal of the resistance of erythrocytes to osmotic lysis was observed. Our findings provide evidence that an oral treatment with vitamin E improves the antioxidant/oxidant balance in plasma, LDL particles, and red blood cells, and counteracts lipid peroxidation processes in beta-thalassemia intermedia patients.

Nonsense-mediated mRNA decay (NMD) is a surveillance system to prevent the synthesis of non-functional proteins. In β-thalassemia, NMD may have a role in clinical outcome. An example of premature translation stop codons appearing for the first time is the β-globin cd39 mutation; when homozygous, this results in a severe phenotype. The aim of this study was to determine whether the homozygous nonsense cd39 may have a milder phenotype in comparison with IVS1,nt110/cd39 genotype. Genotypes have been identified from a cohort of 568 patients affected by β-thalassemia. These genotypes were compared with those found in 577 affected fetuses detected among 2292 prenatal diagnoses. The nine most common genotypes, each with an incidence rate of 1.5% or over, and together accounting for 80% of genotype frequencies, underwent statistical analysis. Genotype prevalence was calculated within the overall group. Results are expressed as proportions with 95% confidence intervals; P≤0.05 was considered statistically significant. A binomial distribution was assumed for each group; z-tests were used to compare genotype frequencies observed in the patient group with frequencies in the affected fetus group. In the absence of selecting factors, prevalence of these two genotypes was compared between a cohort of 568 β-thalassemia patients (PTS) and 577 affected fetuses (FOET) detected during the same period. IVS1,nt110/cd39 was significantly more prevalent in FOET than PTS (P是一种预防非功能性蛋白质合成的监控系统。在β地中海贫血中，NMD可能对临床结果有影响。第一次出现的过早终止密码子（PTC）为β珠蛋白cd39突变；若为纯合子，则会导致严重的表型。本研究旨在确定与IVS1,nt110/cd39基因型相比，纯合子无义cd39能否有更轻度的表型。目前已确定568名β地中海贫血患者的基因型，并与从2292个产前诊断中检测出的577名地中海贫血胎儿的基因型相比较。对9个最常见基因型进行统计分析，每个基因型的发生率均为1.5%或以上，共占基因型频率的80%。在整个组中计算基因型分布情况，其结果以95%置信区间表示；若P≤0.05，则具有统计意义。各组均假定成一个二项分布；Z测试适用于比较患者组的基因型频率和地中海贫血胎儿的基因型频率。 若没有选择因子，则比较568名β地中海贫血患者（PTS）和同一时期所检测到的577个地中海贫血胎儿（FOET）这两组基因型的发生率。IVS1,nt110/cd39在FOET中的发生率明显高于PTS（P

At 5-12 weeks of gestation the amniotic sac is surrounded by celomic fluid, which contains cells of fetal origin. This fluid can be sampled by celocentesis, which involves the ultrasound-guided insertion of a needle through the vagina. The aim of this study was to examine the feasibility of prenatal diagnosis of haemoglobinopathies from the celomic fluid using a specific protocol. Celocentesis was performed at 7-9 weeks gestation in 26 singleton pregnancies at risk for haemoglobinopathies. In 25 cases more than 30 fetal cells were recovered from the celomic fluid and in all these cases molecular analysis for haemoglobinopathies was possible and the results were confirmed by subsequent chorionic villus sampling or amniocentesis. The results of this study suggest that reliable diagnosis of thalassemia syndromes can be performed from 7 weeks gestation by celocentesis. Further work is necessary to demonstrate the safety of celocentesis before widespread use.

Hydroxycarbamide (HC) is a pharmacological agent capable of stimulating fetal haemoglobin (HbF) production during adult life. High levels of HbF may ameliorate the clinical course of β-thalassaemia and sickle cell disease. The efficacy of HC for the treatment of thalassaemia major and thalassaemia intermedia is variable. Although an increase of HbF has been observed in most patients, only some patients experience significant improvement in total haemoglobin levels. This study aimed to determine the effectiveness and safety of short- (1 year) and long-term (mean follow-up 68 months) HC treatment in 24 thalassaemia intermedia patients. Additionally, we evaluated if primary erythroid progenitor cells cultured from treated patients responded to HC treatment in a manner similar to that observed in vivo. Our results confirm a good response to HC after a short-term follow-up in 70% of thalassaemia intermedia patients and a reduction of clinical response in patients with a long follow-up. Erythroid cultures obtained from patients during treatment reproduced the observed in vivo response. Interestingly, haematopoietic stem cells from long-term treated patients showed reduced ability to develop into primary erythroid cultures some months before the reduction of the 'in vivo' response. The mechanism of this loss of response to HC remains to be determined.

The coelomic cavity is part of the extraembryonic mesoderm, surrounding amniotic cavity, embryo, and yolk sac in the early gestation. It is now believed to represent an important transfer interface and a reservoir of nutrients for the embryo. Coelocentesis by ultrasound-guided transvaginal puncture offers an easier access to the early human embryo, from 28 days post-fertilization. However, despite some studies about its biochemical composition being reported, our knowledge about the presence of cellular elements and their quality in this compartment are still limited. Here we studied human coelomic fluids sampled from 6.6 (48 days) to 10 weeks of gestation, demonstrating the presence of functional embryonic erythroid precursors, that is, megaloblasts in the coelomic cavity. The ease of access of the coelomic cavity could allow the development of novel strategies for diagnostic or therapeutic purposes by ultrasound imaging and ultrasound-guided puncture. J Cell Physiol

Background and objective The inherited hemoglobinopathies are a large group of disorders that include thalassemias and hemoglobin variants. Accurate determination of the carrier phenotype is essential for detecting couples at risk for producing offspring with hemoglobinopathy. Heterozygous β-thalassemia is usually silent at the clinical level. His phenotype is characterized by microcytosis and hypochromia with increased hemoglobin A 2 (HbA 2 ) value. Therefore, HbA 2 determination plays a key role in screening programs for hemoglobinopathy. The aim of this review is to address and suggest an approach for reducing or abolishing hemoglobinopathy screening mistakes. Design and methods Quantitative methods for HbA 2 value determination, comment on the accuracy of the test and on the interpretation of data were discussed. The most probable diagnostic conclusion based on the HbA 2 level, hemoglobin pattern, hematological parameters and iron markers was suggested in this review. Results Hemoglobinopathies are the only genetic disease where it is possible to detect carriers using hematological findings rather than DNA analysis. However, hematological diagnosis is sometimes presumptive, and in these cases, DNA analysis becomes necessary. Complete screening is based on the detection of red cell indices, HbA 2 , HbF and hemoglobin variant values. In particular, HbA 2 determination plays a key role in screening programs for β-thalassemia because a small increase in this fraction is one of the most important markers of β-thalassemia heterozygous carriers. Conclusion Genetic factors both related and unrelated to the β- and α-globin gene clusters, iron metabolism, endocrinological disorders, and some types of anemia, together with intra- and inter-laboratory variations in HbA 2 determination, may cause difficulties in evaluating this measurement in screening programs for hemoglobinopathies. Therefore, knowledge of all these issues is important for reducing or eliminating the risk of mistakes in screening programs for hemoglobinopathies.

This study describes a new molecular condition in the alpha(2)-globin gene (HBA2) found in six unrelated families from Southern Italy (Campania and Sicily). This new double mutant form of haemoglobin is called Hb Southern Italy and originated from the coexistence of two known mutations occurring in the same globin gene, HBA2 26 G-->A (Hb Caserta) and HBA2 130 G-->C (Hb Sun Prairie). Hb Sun Prairie was originally observed in Indian patients in either the homozygous state, with severe hemolytic anemia, and in the heterozygous state with microcytosis, or in asymptomatic cases as an alpha-thalassemia carrier phenotype. Hb Caserta was observed for the first time in a Casertian family (South Italy) that displayed a slowmigrating haemoglobin upon investigation. We report the clinical phenotype and molecular study of this new double mutant form of haemoglobin in heterozygous and homozygous subjects, as well as in association with alpha degrees delectional thalassemia.

Although delta-globin gene (HBD MIM#142000) mutations have no clinical implications, co-inheritance of beta- and delta-thalassemia may lead to misdiagnosis. Among 7,153 samples studied for beta-thalassemia, 205 samples with lower than expected HbA2 levels were selected for our analysis and 183 samples (2.5%) were positive for delta-globin gene mutations. Twelve different mutations were detected, and among these five have not been not previously described (HbA2-Catania HBD c.8A--T, HbA2-Corleone HBD c.41C--A, HbA2-Ventimiglia HBD c.212C--G, HbA2-Montechiaro HBD c.260C--A, and HbA2-Bagheria HBD c.422C--T). This study suggests that delta-globin gene defects are very common in Sicily. Thus, these mutations need to be considered during beta-thalassemia screening to avoid false negative results in the detection of at-risk couples.

Hepatocellular carcinoma (HCC) frequently complicates hepatic cirrhosis secondary to viral infection or iron overload. Therefore, patients affected by thalassaemia syndromes have a theoretically high risk of developing the tumour. We collected data on patients attending Italian centres for the treatment of thalassaemia. Twenty-two cases of HCC were identified; 15 were male. At diagnosis, the mean age was 45 +/- 11 years and the mean serum ferritin was 1764 +/- 1448 microg/l. Eighty-six percent had been infected by hepatitis C virus. Nineteen of 22 cases were diagnosed after 1993, suggesting that this problem is becoming more frequent with the aging population of thalassaemia patients.

The natural history of thalassemia has shown substantial change during these years. This applies for each aspect of the pathology (for example, endocrinological, hepatological and psychological) and also for the pathology that has presented and still presents the main cause of death: myocardial dysfunction. In this review, the pathophysiology of cardiac complications, possible role of myocarditis, new knowledge on pathogenesis, and noninvasive detection methods for iron overload in the heart are pointed out. Prophylaxis of cardiomyopathy and new therapy strategies of myocardial dysfunction, including the impact of the new chelation treatment, are discussed.

Introduction: Since HU has been FDA-approved for patients with SCD, it is not clear what proportion are taking a therapeutic dose (≥15mg/kg/day). There is also inadequate reporting of the frequency of HU dose modification during follow-up, or whether those who were treated with subtherapeutic doses ( Methods: Patients were enrolled at the Haematology Department of Ospedale V. Cervello between January 2000 and April 2014. Laboratory parameters and frequency of vaso-occlusive crisis (VOC) and acute chest syndrome (ACS) were recorded. Blood counts, fetal hemoglobin (HbF) response, and changes in SCD complications were compared between the first and last visits to the clinic and across these 3 groups: patients who never took HU were combined with those who suspended HU before the last visit (no HU group, n=50, 36%); HU Results: There were a total of 140 patients: 25 HbSS, 54 HbSβ0thal, and 61 HbSβ+thal. Median follow-up was 6.6 years. The median age was 35 years (range 0.4-61 years). 28% of patients never took HU, and 8% suspended HU treatment during the follow-up. Among patients taking 0.05, Table 1). Similarly, the change in total hemoglobin levels within each group was also insignificant (P all >0.05). HbF decreased in the no HU group, likely due to maturing age of 2 young children included in this group. Both HU treatment groups had modest increases in HbF (P=0.004, 0.001). With respect to SCD complications, the no HU group had less severe disease at the first visit, with lower percent of subjects with and fewer episodes of VOC and ACS (Table 2). While there was an increase in both VOC and ACS with time, this increase was not statistically significant. Both HU treatment groups had a significant reduction in both complications (p Conclusions: About one third of patients with SCD never took or discontinued HU. While these patients may have less severe disease initially, their rates of complications increased during follow-up. Among those taking HU, dose adjustment was common. HU increased HbF and is associated with reducing VOC and ACS. Since the increase in HbF and changes in HU dosing parameters were only modest in patients even on the highest HU doses, titrating HU to the maximum HbF response should be explored in order to improve markers of organ function. Table 1: Hematologic parameters based on HU status First Visit Last Visit NO HU HU 15mg/kg NO HU HU 15mg/kg WBC (k/uL) 11.2 8.74* 10.9 10.7 7.8* 8.3* Hgb (g/dL) 10.0 10.2 10.0 10.2 9.7 9.9 HbF (%) 11.9** 9.4* 10.7* 7.7 11.7* 12.8* *P **includes 4 subjects with hereditary persistent of HbF and 2 children aged 3 months. Table 2: SCD complications based on HU status VOC ACS % of subjects Crisis per patient per year % of subjects Mean episodes per patient F V L V F V L V F V L V F V L V No HU 60 70 2 2.5 22 28 0.2 0.28 HU15mg/kg 96.6 60 4.1* 1.1* 51 25 1.1* 0.32 FV, first visit; LV, last visit *P Disclosures No relevant conflicts of interest to declare.

Orthotopic liver transplantation can be performed successfully in thalassemia. In this article, we describe a case of liver transplantation in a patient with sickle cell/beta-thalassemia complicated by liver sickling. Intrahepatic sickling must be considered in case of allograft dysfunction. This condition can easily be diagnosed by biochemical investigation and liver ultrasonography.

Abstract 2070 Background and Aim. Patients with transfusion-dependent hemoglobinopathies, as thalassemia major (TM) and sickle/β-thalassemia (S/βThal), have an high risk to be infected by hepatitis C virus (HCV), developing chronic hepatitis C (CHC). Current standard-of-care therapy for CHC consists of pegylated interferon-α 2 and ribavirin (PEG-IFN- α/RBV). A genome-wide association study (GWAs) identified a single nucleotide polymorphism (SNP), on chromosome 19, 3 kilobases (kb) upstream of the IL28B gene (IL28B -3kb C>T). This SNP is associated to the natural clearance of HCV-RNA. The IL28B -3kb C>T SNP is in strong linkage disequilibrium with a non-synonymous variant in the exon2 of IL28B gene (213A>G, K70R). Because of in these patients the PEG-IFN-α/RBV treatment of chronic HCV infection could be complicated by severe side adverse events, i.e. ribavirin-associated hemolysis, it is noteworthy to predict the spontaneous C virus clearance by investigate these two IL28B DNA variants. Therefore, in a cohort of 42 patients with hemoglobinopathies, it was analyzed the two mentioned DNA variants to evaluate their allele frequencies and to eventually correlate them to the C virus clearance. Patients and Methods. Forty two patients with hemoglobinopathies, not-treated with PEG- IFN-α/RBV, were included in this study. Molecular analysi:. Genomic DNAs were extracted from peripheral blood mononuclear cells. DNA variants, IL28B -3kb C>T e K70R, were investigated by direct genomic sequencing (CEQ880-Beckman), endonuclease digestion and reverse dot-blot hybridization. PCR primers, specific for the investigated DNA regions, were designed in our laboratory on the basis of IL28B gene map (GENATLAS) and FASTA SNP sequences. According to literature, the genotype IL28B -3kbCC/K70RAA was considered most frequently associated with the spontaneous HCV-RNA clearance, whereas the genotypes IL28B 3kbTT/K70R GG and -3kbCT/K70R AG were considered most frequently associated with a persistent infection. Result: Allele frequencies of -3kbC>T polymorphism were 63,1% for the favourable C allele and 36,9% for the T allele. Allele frequencies for the K70R variant were 63.1% for the A allele and 36,9% for the G allele. Twenty of 42 studied patients (47,6%) showed a spontaneous HCV-RNA clearance: 15/20 (75%) had IL28B genotype -3kbCC/K70R AA, 5/20 (25%) had IL28B genotype -3kbCT/K70R AG. Twenty two of 42 studied patients (52,4%) showed HCV-RNA persistence: 13/22 (59,1%) showed viral genotype 1b and IL28B genotype -3kbCT/K70R AG; 4/22 (18,2%) showed viral genotype 1b and IL28B genotype -3kbCC/K70R AA; 4/22 (18,2%) showed IL28B genotype -3kbTT/K70R GG (3 had viral genotype 2a2c and one viral genotype 1b; one case had viral genotype 2a2c and was homozygous for the favourable -3kb C allele but it was heterozygous for the K70R mutation (Table 1). Conclusion: It seems that IL28B genotype has a marked impact on natural clearance of HCV-RNA: 15/20 (75%) not treated patients who showed a spontaneous HCV-RNA clearance had an IL28B genotype promoting the viral defence (IL28B-3kbCC/K70R AA) (Table 1). These data are in agreement with Thomas et al (Thomas et al. Nature 461,798-801-2009) who showed a -3kb CC genotype in the 60% of patients who clear HCV. Therefore, they may suggest that waiting and watch approach should be advised in patients with hemoglobinopathy and favourable IL28B genotype. Disclosures: No relevant conflicts of interest to declare.

Abstract 5108 Background Hepatocellular carcinoma (HCC) is the most common primary hepatic cancer, and the fifth and eighth most common malignancy worldwide in men and women, respectively. Hepatitis B and C, primary and secondary hemochromatosis and cirrhosis have been identified as major risk factors. Hepatitis C or B virus infection and secondary hemochromatosis are the major cause of morbidity and mortality. In the last years, HCC on cirrhosis is a common reported complication in patients with thalassemia syndromes1,2,3. In non thalassemia patients HCC is a late complication of cirrhosis and prognosis depends on suitability for treatment. In this group, the last is low (40%) considering that cirrhosis severe stage C, according to Child-Pugh score is generally observed (personal communication). Aim of the study describing, in a homogeneous cohort of thalassemia patients, the main clinical findings of HCC. Patients and Methods All subjects with risk factors for HCC as iron overloading, HCV or HBV chronic infection, histological evidence of cirrhosis were undergone every six months to an ultrasound evaluation associated with alphaphetoprotein determination. Nine new cases, since January 2000 until July 2009, were detected. Table I shows the main clinical findings. Mean value of serum ferritin levels, in the last 2 years before diagnosis, was 1049±721. All patients except one were anti-HCV positive (Table I). Seven of them were HCV-RNA positive (Table I). None of them had previous HBV infection. Results Only one patient had severe stage C, according to Child-Pugh score. None of the patients had significant high levels of alphaphetoprotein (AFP) at diagnosis. However, a slightly increase of AFP levels was shown, during the 6 months before diagnosis, in 50% of cases. Three patients had a delayed diagnosis because of atypical imaging for HCC at TC scan or a negative previous histology for HCC after liver biopsy. Five patients had multifocal HCC at the diagnosis (Table I). Four showed an unifocal HCC, developing two of them multifocal HCC during the follow-up (Table I). Four patients died during the follow-up for decompensated cirrhosis. Five patients are alive after treatment. Main used treatments are shown on Table I. The overall mean survival was 28±25 months (range 3-64). Conclusions 1) almost all talassemia patients with HCC are suitable for treatment ; 2) baseline liver function is the most main factor conditioning survival and suitability for treatment ; 3) periodical serum AFP determnations can reveal a slight increase; 3) iron overloading alone can be related to the development of HCC in a liver with still good functional reserve, giving more opportunity for treatment 4) monitoring for HCC by liver ultrasound evaluation, every six months, in at risk patients with thalassemia syndromes should be mandatory. Disclosures No relevant conflicts of interest to declare.

Background: the number of cases with Sickle-Cell Disease (SCD) registered in Italy at 2000 was 928. Six-hundred-twenty-two (67%) of these were with Sickle-Cell/βthalassemia. Moreover, while the main causes of death in Afro-American patients with SCD is well known, we do not have many informations in Italian subjects. For this reason, we carried ahead a case-control study in this group of patients. Aim of the study: the evaluation of the main hematological and clinical parameters predictive of mortality. Materials and Methods: clinical and hematological findings of 14 SCD died patients were compared with a SCD alive control population homogeneous for age, sex, phenotype and number of painful crises per year. Two alive control subjects were studied for each died patient. Results: the main causes of death were: Acute Chest Syndromes (ACS) (n°6), Cardiovascular Diseases (n°3),Hepatic Failure (n°1),Hodgkin Lymphoma (n°1), Stroke (n°1), Lung Cancer (n°1), Surgery Complication (n°1). The main correlated hematological findings with mortality were Hb (gr/dl) levels (p=3/year (Odd Ratio= 1.64; p= Conclusions: these data suggest that the main causes of death in Italian patients with SCD are similar to those reported for Afro-American subjects (M.H. Steinberg, JAMA, April 2, Vol. 289, N°13, 1645–1651). Particularly, the lack of HU treatment, anemia, high ferritin levels, number of crises per year and previous stroke are the most important predictive factors of mortality.

Incidence of Pulmonary Hypertension (PH) has been described in haemoglobinopathic patients, ranging from 10% to 50%, depending on age, therapy, coexisting left ventricular dysfunction (LVD) and classification of the disease. To determine whether PH can occurs in haemoglobinopathic patients in absence of overt LVD, we studied 1121 pts from 7 Thalassemia Centers in Italy(485 Thalassemia Major (TM), 458 thalassemia intermedia (TI), 178 Sickle Cell or Sickle Thalassemia Disease (SCD),49% males, mean age 30 yrs, range 13–56 yrs). In all pts echocardiographic (Echo) defined ejection fraction (EF%) was > 45% and clinically overt LVD was excluded. A cross-sectional Echo study was performed in order to detect PH that was prospectively defined as mild with tricuspid regurgitant jet velocity (TRV)of at least 2.5 m per second and defined as severe with a TRV> 3.5 m per second. Severe PH was observed in 9 pts, 3 with TM, in 6 with TI and in 1 patients with SCD. Mild PH occurs in 46 patients with TM, in 57 patients with TI and 11 pts with SCD. In the remaining patients, PH was in the normal range (TM 89,9%, TI 86,2% and SCD 93,2% of pts, respectively). In the whole group of hemoglobinopathic pts the incidence of PH (mild plus severe) range from 6.5 % to 13.8% of pts. PH (mild to severe) particularly affects pts with TI (13,8% of pts). | Tricuspid Regurgitant Jet Velocity | Thal Major 485 pts | Thal Int 458 pts | SCD 178 pts | % pts | |:----------------------------------:| ------------------ | ---------------- | ----------- | ----- | | TRV3.5 m/sec | 3 (0.6%) | 6 (1.3%) | 1 (0.9%) | 0.9% | Incidence of PH in TM, TI and SCD Our data support a significant presence of PH in haemoglobinopathic pts (6.2 to 13,8%) even though well treated and without LVD. Considering the pathophisiology of haemoglobinopathies a specific consideration to PH in these diseases is required in order to establish a preventive and therapeutic approach.

Sickle cell/ß-Thalassemia is a common disease in areas where ß-Thal and ßS genes are endemic, like in Sicily. In the current study we evaluated clinical and hematological data of Sicilian patients with Sickle cell/ß-Thalassemia treated with Hydroxyurea (HU). The endpoint of the study was to evaluate the efficacy of HU in terms of reduction of sickle cell crises after 2 years of treatment in comparison with the 2 years before. Moreover, we evaluated the outcome after long-term treatment. Fortytwo patients (18 males, mean age 36, range 18–53) were treated with HU (mean dosage 15 mg/kg, range 10–30) for an average 6.6 years follow-up (range 3–9 years). Twentytwo were ß0/ßS and 20 ß+/ßS genotype. All had 3 or more crises in the year before starting HU. We observed a significant reduction in sickle cell crises (7.8 ± 6.9 crises per year versus 0.9 ± 1.8 per year, P < 0.0001), hospitalizations (2.5 ± 2.9 per year versus 0.3 ± 1.5, P < 0.0001), and days in hospital (22.4 ± 21.9 per year versus 1.2 ± 2.3, P < 0.0001). Altogether, there was a 86% reduction in vasoocclusive events in comparison with the 2 years before (P < 0.001). Moreover, there was a significant increase of MCV (71.4 versus 97.5fl, P < 0.0001), HbF (7.5 versus 25.2 %, P < 0.0001), and decrease of WBC (11.4 versus 9.2 109/L P < 0.01) and reticulocytes (14.1 versus 10.2%, P< 0.01). Finally, redution of hyperdense cells and increase of erithropoyetin were seen. After a mean follow-up of 6.6 years, 39 patients are alive. Three died (2 end stage HCV related liver disesases, 1 bleeding after ERCP). Nine of the 40 alive patients developed complications: 1 acute chest syndrome, 2 strokes, 2 myocardial infarctions, 4 bone necrosis. Brain MRI of 15 patients after and during a mean of 6.9 years of HU treatment showed 2 new onset strokes, 1 of which in a patient with a previous stroke. Moreover, 4 patients developed new onset asymptomatic ischemic brain lesions. In every case there had been a significant reduction of sickle cell crises. There were two cases of cancer, occurring in two patients who were brother and sister: lung cancer in the former, a proeviously heavy smoker, breast cancer in the latter. No further serious adverse events were seen. Five patients with iron overload were treated with Deferiprone: no drug interaction with HU was noticed. Our study confirms that HU is effective in reducing clinical relevant crises of patients with Sickle cell/ß-Thalassemia. However, our preliminary data suggest that chronic organ damages are not prevented by HU. Safety has to be assessed by more prolonged studies.

The possibility of increasing Hb F in vivo using drugs like 5-azacytidine, hydroxyurea, and butyrate has been established. However, in many cases this does not entail an increase in total hemoglobin. We report on a patient with Hb Lepore/beta-thalassemia being treated with hydroxyurea (30 mg/Kg/day) because of the presence of erythroid extramedullary masses with severe neurological abnormalities. During therapy the patient showed a remarkable improvement in neurological signs due to the reduction in extra-medullary masses, a significant increase in both total hemoglobin (from 5.8 to 9.7 g/dl) and Hb F (from 4.9 g/dl to 9.1 g/dl). The marked improvement in hemoglobin level in our patient with Hb Lepore/beta-thalassemia suggests gamma-globin gene activation due to the DNA structure determined by the crossover event.