Incidence of cancer and related deaths in hemoglobinopathies: A follow-up of 4631 patients between 1970 and 2021.

BACKGROUND: The correlation between thalassemia and malignancies other than hepatocellular carcinoma (HCC) and the possible relationship between other hemoglobinopathies and tumor risk have been poorly evaluated. METHODS: Eight Italian specialized centers evaluated the incidence of malignant neoplasms in hemoglobinopathies as well as their sites and features. The study cohort included 4631 patients followed between 1970 and 2021 (transfusion-dependent ß-thalassemia, 55.6%; non-transfusion-dependent thalassemia, 17.7%; sickle cell disease, 17.6%; hemoglobin H disease, 8.3%). RESULTS: A total of 197 diagnoses of cancer were reported (incidence rate, 442 cases per 100,000 person-years). The liver was the most frequent site of tumors in both sexes, with a higher incidence (190 cases per 100,000 person-years) in comparison with the general population found in all types of hemoglobinopathies (except hemoglobin H disease). In recent years, tumors have become the second cause of death in patients with transfusion-dependent thalassemia. A lower risk of breast and prostate cancer was observed in the whole group of patients with hemoglobinopathies. The first cancer diagnoses dated back to the 1980s, and the incidence rate sharply increased after the 2000s. However, although the incidence rate of cancers of all sites but the liver continued to show an increasing trend, the incidence of HCC showed stability. CONCLUSIONS: These findings provide novel insights into the relationship between cancer and hemoglobinopathies and suggest that the overall risk is not increased in these patients. HCC has been confirmed as the most frequent tumor, but advances in chelation and the drugs that have led to the eradication of hepatitis C may explain the recent steadiness in the number of diagnoses that is reported here.

Deep Learning Staging of Liver Iron Content From Multiecho MR Images.

BACKGROUND: MRI represents the most established liver iron content (LIC) evaluation approach by estimation of liver T2* value, but it is dependent on the choice of the measurement region and the software used for image analysis. PURPOSE: To develop a deep-learning method for unsupervised classification of LIC from magnitude T2* multiecho MR images. STUDY TYPE: Retrospective. POPULATION/SUBJECTS: A total of 1069 thalassemia major patients enrolled in the core laboratory of the Myocardial Iron Overload in Thalassemia (MIOT) network, which were included in the training (80%) and test (20%) sets. Twenty patients from different MRI vendors included in the external test set. FIELD STRENGTH/SEQUENCE: A5 T, T2* multiecho magnitude images. ASSESSMENT: Four deep-learning convolutional neural networks (HippoNet-2D, HippoNet-3D, HippoNet-LSTM, and an ensemble network HippoNet-Ensemble) were used to achieve unsupervised staging of LIC using five classes (normal, borderline, middle, moderate, severe). The training set was employed to construct the deep-learning model. The performance of the LIC staging model was evaluated in the test set and in the external test set. The model's performances were assessed by evaluating the accuracy, sensitivity, and specificity with respect to the ground truth labels obtained by T2* measurements and by comparison with operator-induced variability originating from different region of interest (ROI) placements. STATISTICAL TESTS: The network's performances were evaluated by single-class accuracy, specificity, and sensitivity and compared by one-way repeated measures analysis of variance (ANOVA) and one-way ANOVA. RESULTS: HippoNet-Ensemble reached an accuracy significantly higher than the other networks, and a sensitivity and specificity higher than HippoNet-LSTM. Accuracy, sensitivity, and specificity values for the LIC stages were: normal: 0.96/0.93/0.97, borderline: 0.95/0.85/0.98, mild: 0.96/0.88/0.98, moderate: 0.95/0.89/0.97, severe: 0.97/0.95/0.98. Correctly staging of cases was in the range of 85%-95%, depending on the LIC class. Multiclass accuracy was 0.90 against 0.92 for the interobserver variability. DATA CONCLUSION: The proposed HippoNet-Ensemble network can perform unsupervised LIC staging and achieves good prognostic performance. EVIDENCE LEVEL: 4 TECHNICAL EFFICACY: Stage 2.

Overall and complication-free survival in a large cohort of patients with ß-thalassemia major followed over 50 years.

We report data on survival and complications for a longitudinal cohort of 709 transfusion-dependent ß-thalassemia major patients (51.1% males) born between 1970 and 1997 and followed through 2020 at seven major centers in Italy. Overall survival probability at 30 years was 83.6% (95%CI: 78.5-89.1) in the oldest birth cohort (1970-1974) compared with 93.3% (95%CI: 88.6-98.3) in the youngest birth cohort (1985-1997) (p = 0.073). Females showed better survival than males (p = 0.022). There were a total of 93 deaths at a median age of 23.2 years with the most frequent disease-related causes being heart disease (n = 53), bone marrow transplant (BMT) complication (n = 10), infection (n = 8), liver disease (n = 4), cancer (n = 3), thromboembolism (n = 2) and severe anemia (n = 1). There was a steady decline in the number of deaths due to heart disease from the year 2000 onwards and no death from BMT was observed after the year 2010. A progressive decrease in the median age of BMT was observed in younger birth cohorts (p < 0.001). A total of 480 (67.7%) patients developed ≥1 complication. Patients in younger birth cohorts demonstrated better complication-free survival (p < 0.001) which was comparable between sexes (p = 0.230). Independent risk factors for death in multivariate analysis included heart disease (HR: 4.63, 95%CI: 1.78-12.1, p = 0.002), serum ferritin >1000 ng/mL (HR: 15.5, 95%CI: 3.52-68.2, p < 0.001), male sex (HR: 2.75, 95%CI: 0.89-8.45, p = 0.078), and splenectomy (HR: 6.97, 95%CI: 0.90-54.0, p < 0.063). Survival in patients with ß-thalassemia major continues to improve with adequate access to care, best practice sharing, continued research, and collaboration between centers.

National networking in rare diseases and reduction of cardiac burden in thalassemia major.

AIMS: A tailored chelation therapy guided by magnetic resonance imaging (MRI) is a strategy to improve the prognosis in iron-loaded patients, in many cases still hampered by limited MRI availability. In order to address this issue, the Myocardial Iron Overload in Thalassemia (MIOT) network was established in Italy and we aimed to describe the impact of 10-year activity of this network on cardiac burden in thalassemia major (TM). METHODS AND RESULTS: Within the MIOT network, 1746 TM patients (911 females; mean age 31.2 ± 9.1 years) were consecutively enrolled and prospectively followed by 70 thalassemia and 10 MRI centres. Patients were scanned using a multiparametric approach for assessing myocardial iron overload (MIO), biventricular function, and myocardial fibrosis. At the last MRI scan, a significant increase in global heart T2* values and a significantly higher frequency of patients with no MIO (all segmental T2* ≥20 ms) were detected, with a concordant improvement in biventricular function, particularly in patients with baseline global heart T2* <20 ms. Forty-seven percentage of patients changed the chelation regimen based on MRI. The frequency of heart failure (HF) significantly decreased after baseline MRI from 3.5 to 0.8% (P < 0.0001). Forty-six patients died during the study, and HF accounted for 34.8% of deaths. CONCLUSION: Over 10 years, continuous monitoring of cardiac iron and a tailored chelation therapy allowed MIO reduction, with consequent improvement of cardiac function and reduction of cardiac complications and mortality from MIO-related HF. A national networking for rare diseases therefore proved effective in improving the care and reducing cardiac outcomes of TM patients.

Decrease in α-Globin and Increase in the Autophagy-Activating Kinase ULK1 mRNA in Erythroid Precursors from ß-Thalassemia Patients Treated with Sirolimus.

The ß-thalassemias are hereditary monogenic diseases characterized by a low or absent production of adult hemoglobin and excess in the content of α-globin. This excess is cytotoxic for the erythroid cells and responsible for the ß-thalassemia-associated ineffective erythropoiesis. Therefore, the decrease in excess α-globin is a relevant clinical effect for these patients and can be realized through the induction of fetal hemoglobin, autophagy, or both. The in vivo effects of sirolimus (rapamycin) and analogs on the induction of fetal hemoglobin (HbF) are of key importance for therapeutic protocols in a variety of hemoglobinopathies, including ß-thalassemias. In this research communication, we report data showing that a decrease in autophagy-associated p62 protein, increased expression of ULK-1, and reduction in excess α-globin are occurring in erythroid precursors (ErPCs) stimulated in vitro with low dosages of sirolimus. In addition, increased ULK-1 mRNA content and a decrease in α-globin content were found in ErPCs isolated from ß-thalassemia patients recruited for the NCT03877809 clinical trial and treated with 0.5-2 mg/day sirolimus. Our data support the concept that autophagy, ULK1 expression, and α-globin chain reduction should be considered important endpoints in sirolimus-based clinical trials for ß-thalassemias.

Thalassaemia is paradoxically associated with a reduced risk of in-hospital complications and mortality in COVID-19: Data from an international registry.

Although numerous patient-specific co-factors have been shown to be associated with worse outcomes in COVID-19, the prognostic value of thalassaemic syndromes in COVID-19 patients remains poorly understood. We studied the outcomes of 137 COVID-19 patients with a history of transfusion-dependent thalassaemia (TDT) and transfusion independent thalassaemia (TIT) extracted from a large international cohort and compared them with the outcomes from a matched cohort of COVID-19 patients with no history of thalassaemia. The mean age of thalassaemia patients included in our study was 41 ± 16 years (48.9% male). Almost 81% of these patients suffered from TDT requiring blood transfusions on a regular basis. 38.7% of patients were blood group O. Cardiac iron overload was documented in 6.8% of study patients, whereas liver iron overload was documented in 35% of study patients. 40% of thalassaemia patients had a history of splenectomy. 27.7% of study patients required hospitalization due to COVID-19 infection. Amongst the hospitalized patients, one patient died (0.7%) and one patient required intubation. Continuous positive airway pressure (CPAP) was required in almost 5% of study patients. After adjustment for age-, sex- and other known risk factors (cardiac disease, kidney disease and pulmonary disease), the rate of in-hospital complications (supplemental oxygen use, admission to an intensive care unit for CPAP therapy or intubation) and all-cause mortality was significantly lower in the thalassaemia group compared to the matched cohort with no history of thalassaemia. Amongst thalassaemia patients in general, the TIT group exhibited a higher rate of hospitalization compared to the TDT group (p = 0.001). In addition, the rate of complications such as acute kidney injury and need for supplemental oxygen was significantly higher in the TIT group compared to the TDT group. In the multivariable logistic regression analysis, age and history of heart or kidney disease were all found to be independent risk factors for increased in-hospital, all-cause mortality, whereas the presence of thalassaemia (either TDT or TIT) was found to be independently associated with reduced all-cause mortality. The presence of thalassaemia in COVID-19 patients was independently associated with lower in-hospital, all-cause mortality and few in-hospital complications in our study. The pathophysiology of this is unclear and needs to be studied in vitro and in animal models.

Beta-thalassaemia major: Prevalence, risk factors and clinical consequences of hypercalciuria.

Regular transfusion and chelation therapy produces increased life expectancy in thalassaemic patients who may develop new complications. Since few data are available regarding hypercalciuria in ß-thalassaemia major (TM), the aim of our study was to evaluate its prevalence, risk factors and clinical consequences. We enrolled 176 adult TM patients followed at the Center of Thalassemia of Ferrara. Hypercalciuria was defined by a calciuria of 4 mg/kg/day or more in a 24-h urine sample. Anamnestic, biochemical and radiological data were collected. Hypercalciuria prevalence was reported in 69.3% of patients (females 52.5%). Hypercalciuric (HC) patients used deferasirox (DFX) more often than normocalciuric (NC) patients (47.5% vs 29.6%; p < 0.05). In HC subjects plasma parathyroid hormone (PTH) (24.1 ± 10.4 vs 30.1 ± 13.2 pg/ml) and phosphate levels (3.6 ± 0.5 vs 3.8 ± 0.7 mg/dl) were lower, whereas serum calcium (9.6 ± 0.4 vs 9.4 ± 0.4 mg/dl) and urinary 24-h phosphaturia (0.9 ± 0.4 vs 0.6 ± 0.3 g/day) were higher as compared to NC patients (p < 0.05 for all comparisons). Supplementation with oral calcium and cholecalciferol was similar between the groups. A higher rate of kidney stones was present in HC (14.8%) versus NC patients (3.7%) (p < 0.05). Hypercalciuria is a frequent complication in adequately treated adult TM patients. Hypercalciuria prevalence is increased in DFX users whereas haemoglobin level or calcium supplements play no role. A significant proportion of HC patients developed kidney stones.

Efficacy and Safety of Teriparatide in Beta-Thalassemia Major Associated Osteoporosis: A Real-Life Experience.

Osteoporosis represents a relevant cause of morbidity in adult Thalassemia Major (TM) population. Antiresorptive drugs such as bisphosphonates were demonstrated effective in preventing bone loss. Teriparatide (TP) is an anabolic agent approved for osteoporosis management in the general population, but its use has been very limited in TM patients so far. We evaluated TP efficacy and safety in TM-associated osteoporosis in real-life clinical practice. Retrospective evaluation of 11 TM patients (6 males, 5 females; mean age = 45 ± 4.38 years) with severe osteoporosis and multiple fractures under TP treatment. Mean TP treatment duration was 19 ± 7 months. TP withdrawal was due to poor compliance and side effects (fever and osteo-muscular pain) in two and three patients, respectively. After 12 and 24 months, BMD significantly increased at lumbar (+ 19% and 22%) and femoral sites (+ 13% and 13%). Osteocalcin and cross-laps levels increased after 12 and 24 months (+ 225 and + 54.2%; + 159 and 141%, respectively). No new fractures were detected during TP treatment. Baseline VAS score values (3 ± 3) did not significantly change after 12 and 24 months (3 ± 3 and 2 ± 3, respectively). Five out of eleven patients developed side effects. TP might be an effective treatment for TM-associated osteoporosis since it improves BMD, especially at the lumbar spine, and prevents fragility fractures. TM patients may have a higher frequency of side effects, especially muscle and bone pain under TP treatment, as compared to no TM population. Further studies are needed.

The role of opportunistic quantitative computed tomography in the evaluation of bone disease and risk of fracture in thalassemia major.

OBJECTIVE: Dual-energy X-ray absorptiometry (DXA) remains the cornerstone for osteoporosis evaluation in Thalassemia major. However, several drawbacks have been observed in this unique setting. We sought to determine the correlation between quantitative CT (QCT) and DXA-derived parameters; secondarily, we aimed to investigate the role of the two techniques in predicting the risk of fracture. METHODS: We retrospectively included patients with ß-thalassemia major who had undergone both lumbar and femoral DXA examinations, and CT scans including the lumbar spine, performed for disparate diagnostic issues, within 4 months from the DXA. CT data were examined employing a phantom-less QCT method for bone mineral density (BMD) assessment. We also retrieved any spontaneous or fragility fractures occurring from 1 year before up to 5 years after the date of DXA scans. RESULTS: The 43 patients were included. QCT measures were significantly higher than those determined by DXA. The gap between QCT and DXA values was strongly associated with patient age. The most powerful predictive variable for risk of fracture was the ACR classification based on volumetric BMD obtained by QCT. CONCLUSIONS: DXA provided more negative measures than those determined by QCT. However, QCT seemed to evaluate thalassaemic osteopathy better than DXA, since volumetric BMD was a stronger predictor of fracture.

Relationship between pancreatic iron overload, glucose metabolism and cardiac complications in sickle cell disease: An Italian multicentre study.

OBJECTIVES: Evidence about the cross-talk between iron, glucose metabolism, and cardiac disease is increasing. We aimed to explore the link of pancreatic iron by Magnetic Resonance Imaging (MRI) with glucose metabolism and cardiac complications (CC) in sickle cell disease (SCD) patients. METHODS: We considered 70 SCD patients consecutively enrolled in the Extension-Myocardial Iron Overload in Thalassemia Network. Iron overload was quantified by R2* technique and biventricular function by cine images. Macroscopic myocardial fibrosis was evaluated by late gadolinium enhancement technique. Glucose metabolism was assessed by the oral glucose tolerance test. RESULTS: Patients with an altered glucose metabolism showed a significantly higher pancreas R2* than patients with normal glucose metabolism. Pancreatic siderosis emerged as a risk factor for the development of metabolic alterations (OddsRatio 8.25, 95%confidence intervals 1.51-45.1; p = .015). Global pancreas R2* values were directly correlated with mean serum ferritin levels and liver iron concentration. Global pancreas R2* was not significantly associated with global heart R2* and macroscopic myocardial fibrosis. Patients with history of CC showed a significantly higher global pancreas R2* than patients with no CC. CONCLUSIONS: Our findings support the evaluation of pancreatic R2* by MRI in SCD patients to prevent the development of metabolic and cardiac disorders.

Luspatercept improves hemoglobin levels and blood transfusion requirements in a study of patients with ß-thalassemia.

ß-thalassemia is a hereditary disorder with limited approved treatment options; patients experience anemia and its complications, including iron overload. The study aim was to determine whether luspatercept could improve anemia and disease complications in patients with ß-thalassemia. This open-label, nonrandomized, uncontrolled study consisted of a 24-week dose-finding and expansion stage (initial stage) and a 5-year extension stage, currently ongoing. Sixty-four patients were enrolled; 33 were non-transfusion dependent (mean hemoglobin, <10.0 g/dL; <4 red blood cell [RBC] units transfused per 8 weeks), and 31 were transfusion dependent (≥4 RBC units per 8 weeks). Patients received 0.2 to 1.25 mg/kg luspatercept subcutaneously every 21 days for ≥5 cycles (dose-finding stage) and 0.8 to 1.25 mg/kg (expansion cohort and 5-year extension). The primary end point was erythroid response, defined as hemoglobin increase of ≥1.5 g/dL from baseline for ≥14 consecutive days (without RBC transfusions) for non-transfusion-dependent patients or RBC transfusion burden reduction ≥20% over a 12-week period vs the 12 weeks before treatment for transfusion-dependent patients. Eighteen non-transfusion-dependent patients (58%) receiving higher dose levels of luspatercept (0.6-1.25 mg/kg) achieved mean hemoglobin increase ≥1.5 g/dL over ≥14 days vs baseline. Twenty-six (81%) transfusion-dependent patients achieved ≥20% reduction in RBC transfusion burden. The most common grade 1 to 2 adverse events were bone pain, headache, and myalgia. As of the cutoff, 33 patients remain on study. In this study, a high percentage of ß-thalassemia patients receiving luspatercept had hemoglobin or transfusion burden improvements. These findings support a randomized clinical trial to assess efficacy and safety. This study was registered at www.clinicaltrials.gov as #NCT01749540 and #NCT02268409.

Biventricular Reference Values by Body Surface Area, Age, and Gender in a Large Cohort of Well-Treated Thalassemia Major Patients Without Heart Damage Using a Multiparametric CMR Approach.

BACKGROUND: Cardiac MRI plays a critical role in the management of thalassemic patients. No accurate biventricular reference values are available. PURPOSE: To establish the ranges for normal left ventricular (LV) and right ventricular (RV) volumes and ejection fraction (EF) and LV mass normalized to body surface area (BSA), age, and gender in a large cohort of well-treated beta-thalassemia major (ß-TM) patients without heart damage using a multiparametric MRI. STUDY TYPE: Retrospective/cohort study. POPULATION: In all, 251 ß-TM patients with no known risk factors or cardiac disease, normal electrocardiogram, no macroscopic myocardial fibrosis, and all cardiac segments with T2 * ≥20 msec, and 246 healthy subjects. FIELD STRENGTH/SEQUENCE: 1.5T/cine steady-state free precession (SSFP), gradient-echo T2 *, late gadolinium enhancement (LGE) images. ASSESSMENT: Biventricular end-diastolic volume, end-systolic volume, stroke volume, and LV mass were normalized to BSA (EDVI, ESVI, SVI). STATISTICAL TESTS: Comparisons between the two groups was performed with two-samples t-test or Wilcoxon's signed rank test. For more than two groups, one-way analysis of variance (ANOVA) or a Kruskal-Wallis test were applied. RESULTS: Compared to controls, males with ß-TM showed significantlt higher LVEDVI in all the age groups, while for the other volumes the difference was significant only within one or more age groups. In females the volumes were comparable between ß-TM patients and healthy subjects in all the age groups. In the male ß-TM population we found a significant effect of age on LVEDVI (P = 0.017), LVESVI (P = 0.001), RVESVI (P = 0.029), and RVEF (P = 0.031), while for females none of the biventricular parameters were significantly different among the age groups (LVEDVI: P = 0.614; LVESVI: P = 0.449; LVSVI: P = 0.186; LV mass index: P = 0.071; LVEF: P = 0.059; RVEDVI: P = 0.374; RVESVI: P = 0.180; RVSVI: P = 0.206; RVEF: P = 0.057). In ß-TM patients all biventricular volume indexes as well as the LV mass index were significantly larger in males than in females (P < 0.0001 in all cases). The LV and the RV EF were comparable between the sexes (P = 0.568 and P = 0.268, respectively). DATA CONCLUSION: Appropriate "normal" reference ranges normalized to BSA, sex, and age are recommended to avoid misdiagnosis of cardiomyopathy in ß-TM patients. LEVEL OF EVIDENCE: 4 TECHNICAL EFFICACY STAGE: 2.

Impact of the preparation method of red cell concentrates on transfusion indices in thalassemia patients: A randomized crossover clinical trial.

BACKGROUND: The average hemoglobin content of red cell concentrates (RCC) varies depending on the method of preparation. Surprisingly less data are available concerning the clinical impact of those differences. STUDY DESIGN AND METHODS: The effects of two types of RCC (RCC-A, RCC-B) on transfusion regime were compared in a non-blinded, prospective, randomized, two-period, and crossover clinical trial. RCC-A was obtained by whole blood leukoreduction and subsequent plasma removal, RCC-B removing plasma and buffy coat first, followed by leukoreduction. Eligible patients were adult, with transfusion-dependent thalassemia (TDT). RESULTS: RCC-A contained 63.9 (60.3-67.8) grams of hemoglobin per unit (median with 1st and 3rd quartile), RCC-B 54.5 (51.0-58.2) g/unit. Fifty-one patients completed the study. With RCC-B, the average pre-transfusion hemoglobin concentration was 9.3 ± 0.5 g/dl (mean ± SD), the average transfusion interval 14.2 (13.7-16.3) days, the number of RCC units transfused per year 39.3 (35.4-47.3), and the transfusion power index (a composite index) 258 ± 49. With RCC-A, the average pre-transfusion hemoglobin concentration was 9.6 ± 0.5 g/dl (+2.7%, effect size 0.792), the average transfusion interval 14.8 (14.0-18.5) days (+4.1%, effect size 0.800), the number of RCC units transfused per year 34.8 (32.1-42.5) (-11.4%, effect size -1.609), and the transfusion power index 272 ± 61 (+14.1%, effect size 0.997). All differences were statistically highly significant (p < .00001). The frequency of transfusion reactions was 0.59% with RCC-A and 0.56% with RCC-B (p = 1.000). CONCLUSION: To reduce the number of RCC units consumed per year and the number of transfusion episodes, TDT patients should receive RCC with the highest average hemoglobin content.

The effect of desferrioxamine chelation versus no therapy in patients with non transfusion-dependent thalassaemia: a multicenter prospective comparison from the MIOT network.

We prospectively assessed by magnetic resonance imaging (MRI) the advantages of desferrioxamine (DFO) with respect to the absence of chelation therapy in non transfusion-dependent thalassaemia (NTDT) patients. We considered 18 patients non-chelated and 33 patients who received DFO alone between the two MRI scans. Iron overload was assessed by the T2* technique. Biventricular function parameters were quantified by cine sequences. No patient treated with DFO had cardiac iron. At baseline, only one non-chelated patient showed a pathological heart T2* value (< 20 ms) and he recovered at the follow-up. The percentage of patients who maintained a normal heart T2* value was 100% in both groups. A significant increase in the right ventricular ejection fraction was detected in DFO patients (3.48 ± 7.22%; P = 0.024). The changes in cardiac T2* values and in the biventricular function were comparable between the two groups. In patients with hepatic iron at baseline (MRI liver iron concentration (LIC) ≥ 3 mg/g/dw), the reduction in MRI LIC values was significant only in the DFO group (- 2.20 ± 4.84 mg/g/dw; P = 0.050). The decrease in MRI LIC was comparable between the groups. In conclusion, in NTDT patients, DFO therapy showed no advantage in terms of cardiac iron but its administration allowed an improvement in right ventricular function. Moreover, DFO reduced hepatic iron in patients with significant iron burden at baseline.

An Aγ-globin G->A gene polymorphism associated with ß039 thalassemia globin gene and high fetal hemoglobin production.

BACKGROUND: Increase of the expression of γ-globin gene and high production of fetal hemoglobin (HbF) in ß-thalassemia patients is widely accepted as associated with a milder or even asymptomatic disease. The search for HbF-associated polymorphisms (such as the XmnI, BCL11A and MYB polymorphisms) has recently gained great attention, in order to stratify ß-thalassemia patients with respect to expectancy of the first transfusion, need for annual intake of blood, response to HbF inducers (the most studied of which is hydroxyurea). METHODS: Aγ-globin gene sequencing was performed on genomic DNA isolated from a total of 75 ß-thalassemia patients, including 31 ß039/ß039, 33 ß039/ß+IVSI-110, 9 ß+IVSI-110/ß+IVSI-110, one ß0IVSI-1/ß+IVSI-6 and one ß039/ß+IVSI-6. RESULTS: The results show that the rs368698783 polymorphism is present in ß-thalassemia patients in the 5'UTR sequence (+25) of the Aγ-globin gene, known to affect the LYAR (human homologue of mouse Ly-1 antibody reactive clone) binding site 5'-GGTTAT-3'. This Aγ(+25 G->A) polymorphism is associated with the Gγ-globin-XmnI polymorphism and both are linked with the ß039-globin gene, but not with the ß+IVSI-110-globin gene. In agreement with the expectation that this mutation alters the LYAR binding activity, we found that the Aγ(+25 G->A) and Gγ-globin-XmnI polymorphisms are associated with high HbF in erythroid precursor cells isolated from ß039/ß039 thalassemia patients. CONCLUSIONS: As a potential explanation of our findings, we hypothesize that in ß-thalassemia the Gγ-globin-XmnI/Aγ-globin-(G->A) genotype is frequently under genetic linkage with ß0-thalassemia mutations, but not with the ß+-thalassemia mutation here studied (i.e. ß+IVSI-110) and that this genetic combination has been selected within the population of ß0-thalassemia patients, due to functional association with high HbF. Here we describe the characterization of the rs368698783 (+25 G->A) polymorphism of the Aγ-globin gene associated in ß039 thalassemia patients with high HbF in erythroid precursor cells.

A validated cellular biobank for ß-thalassemia.

BACKGROUND: Cellular biobanking is a key resource for collaborative networks planning to use same cells in studies aimed at solving a variety of biological and biomedical issues. This approach is of great importance in studies on ß-thalassemia, since the recruitment of patients and collection of specimens can represent a crucial and often limiting factor in the experimental planning. METHODS: Erythroid precursor cells were obtained from 72 patients, mostly ß-thalassemic, expanded and cryopreserved. Expression of globin genes was analyzed by real time RT-qPCR. Hemoglobin production was studied by HPLC. RESULTS: In this paper we describe the production and validation of a Thal-Biobank constituted by expanded erythroid precursor cells from ß-thalassemia patients. The biobanked samples were validated for maintenance of their phenotype after (a) cell isolation from same patients during independent phlebotomies, (b) freezing step in different biobanked cryovials, (c) thawing step and analysis at different time points. Reproducibility was confirmed by shipping the frozen biobanked cells to different laboratories, where the cells were thawed, cultured and analyzed using the same standardized procedures. The biobanked cells were stratified on the basis of their baseline level of fetal hemoglobin production and exposed to fetal hemoglobin inducers. CONCLUSION: The use of biobanked cells allows stratification of the patients with respect to fetal hemoglobin production and can be used for determining the response to the fetal hemoglobin inducer hydroxyurea and to gene therapy protocols with reproducible results.