Chronic Administration of Hydroxyurea (HU) Benefits Caucasian Patients with Sickle-Beta Thalassemia.

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 (<15 mg/kg/day) are beneficial. We reviewed the medical records of 140 patients from 2010 to 2014. The laboratory parameters and SCD complications were compared between the first and last visits based on HU use. Fifty patients (36%) never took HU or suspended HU ("no HU" group). Among patients taking <15 mg/kg/day HU on their first visit, half remained at the same dose, and the other half increased to ≥15 mg/kg/day. Among patients taking ≥15 mg/kg/day, 17% decreased to <15 mg/kg/day, and 83% stayed at ≥15 mg/kg/day. The "no HU" group had fewer episodes of VOC and ACS. Both HU treatment groups had a reduction in both complications (p < 0.0001). This improvement was observed in all SCD phenotypes. The white blood cell (WBC) counts were found to be lower, and HbF increased in both HU groups (p = 0.004, 0.001). The maximal HbF response to HU in HbS/ß⁺-thalassemia was 20%, similar to those observed for HbSS (19%) and HbS/ß°-thalassemia (22%). HbS/ß⁺-thalassemia could have a similar disease severity as HbSS or HbS/ß°-thalassemia. Patients with HbS/ß°-thalassemia or HbS/ß⁺-thalassemia phenotypes responded to HU.

HBB: c.316-125A>G and HBB: c.316-42delC: Phenotypic Evaluations of Two Rare Changes in the Second Intron of the HBB Gene.

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) (ß+), NM_000518, HBB: c.316-125A>G] and a deletion of a cytosine at nt 809 [IVS-II-809 (-C) (ß), 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 ß-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.

Embryo-fetal erythroid cell selection from celomic fluid allows earlier prenatal diagnosis of hemoglobinopathies.

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 (< 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 was > 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.

Deferiprone versus deferoxamine in sickle cell disease: results from a 5-year long-term Italian multi-center randomized clinical trial.

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.

Feasibility of DNA diagnosis of haemoglobinopathies on coelocentesis.

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.

The genetic heterogeneity of ß-globin gene defects in Sicily reflects the historic population migrations of the island.

The aim of this study is to update the incidence and the distribution of the globin gene defects causing ß-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 ß-thalassemia defects accounted for 95.11% of examined alleles while other beta-globin gene defects were found at lower frequencies (<1%). A consistent number (24) of variant hemoglobins were identified of whom Hb S was the most represented (72.1%). Our data underline the heterogeneity of the beta-globin gene defects in the Sicily. The enormous progress in the technique for ß-globin gene analysis permitted to characterize 99.93% of mutated alleles and it has made a first trimester prenatal diagnosis program possible in our region in all cases with a great improvement in thalassemia management. The origin of the large spectrum of mutations is discussed taking in consideration the history of the island.

Embryo-fetal erythroid megaloblasts in the human coelomic cavity.

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.

Desensitization to hydroxycarbamide following long-term treatment of thalassaemia intermedia as observed in vivo and in primary erythroid cultures from treated patients.

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 ß-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.

Hb Southern Italy: coexistence of two missence mutations (the Hb Sun Prairie alpha2 130 Ala --> Pro and Hb Caserta alpha2 26 Ala --> Thr) in a single HBA2 gene.

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.

Significance of borderline hemoglobin A2 values in an Italian population with a high prevalence of beta-thalassemia.

We report a retrospective analysis carried out on 23,485 subjects submitted to a screening program from 2000 to 2006. Of these subjects, 3,934 had borderline HbA(2) values from 3.1 to 3.9%; 410 samples, analyzed previously using PCR methods and sequencing because all of these were partners of a carrier of classical beta-thalassemia, were selected for statistical analysis. Of 410 subjects, 94 (22.9%) were positive for a molecular defect in the beta-, delta- or alpha-globin genes. The most prevalent molecular defects were beta IVS1 nt 6 (HBB c.92+6T C), co-inheritance of severe beta thalassemia and delta mutations, beta-promoter mutations and triplication of alpha genes were detected; alpha-thalassemia and Hb-variants were also evident. Borderline HbA(2) is not a rare event in a population with a high prevalence of beta-thalassemia carriers. These data support the necessity to investigate these cases at a molecular level, particularly if the partner is a carrier of beta-thalassemia.

ß-Thalassemia heterozygote state detrimentally affects health expectation.

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 ß-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.

Phenotypic evaluations of HBB:c.93-23T>C, a nucleotide substitution in the IVS I nt 108 of ß-globin gene.

BACKGROUND: Thalassaemia and variant haemoglobin are the most common severe monogenic disorders worldwide. AIMS: To develop prenatal diagnosis programmes for the prevention of the most important haemoglobin disorders and identify healthy carriers of thalassaemia. METHODS: Sequencing 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. RESULTS: A 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. CONCLUSION: This 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 ß-thalassaemia and the other is carrier of a nucleotide variation.

Analysis of delta-globin gene alleles in the Sicilian population: identification of five new mutations.

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.

Cardiac complications in thalassemia: noninvasive detection methods and new directions in the clinical management.

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.

The significance of the hemoglobin A(2) value in screening for hemoglobinopathies.

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 beta-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 beta-thalassemia because a small increase in this fraction is one of the most important markers of beta-thalassemia heterozygous carriers. CONCLUSION: Genetic factors both related and unrelated to the beta- and alpha-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.

Hepatocellular carcinoma in the thalassaemia syndromes.

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.