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Initiation of regular transfusion in transfusion-dependent thalassemia (TDT) is based on the assessment of clinical phenotype. Pathogenic HBB variants causing β-thalassemia are important determinants of phenotype and could be used to aid decision-making. We investigated the association of HBB genotype with survival in a cohort study in the four thalassemia centers in Cyprus. HBB genotype was classified as severe (β0/β0 or β+/β0), moderate (β+/β+), or mild (β0/β++ or β+/β++). Risk factors for mortality were evaluated using multivariate Cox proportional- hazards regression. Of the 537 subjects who were followed for a total of 20,963 person-years, 80.4% (95% confidence interval [95% CI]: 76.4-84.7) survived to 50 years of age with increasing rates of liver-, infectionand malignancy-related deaths observed during recent follow-up. We evaluated non-modifiable risk factors and found worse outcomes associated with male sex (hazard ratio 1.9, 95% CI: 1.1-3.0, P=0.01) and milder genotype (hazard ratio 1.6, 95% CI: 1.1-2.3, P=0.02). The effect of genotype was confirmed in a second model, which included treatment effects. Patients with a milder genotype initiated transfusion significantly later and had reduced blood requirements compared to those with moderate or severe genotypes, although pre-transfusion hemoglobin levels did not differ between genotypes. Our results suggest that early treatment decisions to delay transfusion and different long-term treatment strategies in individuals with milder genotypes have led to adverse longterm effects of under-treated thalassemia and worse survival. We propose that HBB genotype determination and use of this information to aid in decision-making can improve long-term outcomes of thalassemia patients.

β-thalassemia is a disorder caused by altered hemoglobin protein synthesis and affects individuals worldwide. Severe forms of the disease, left untreated, can result in death before the age of 3 years (1). The standard of care consists of chronic and costly palliative treatment by blood transfusion combined with iron chelation. This dual approach suppresses anemia and reduces iron-related toxicities in patients. Allogeneic bone marrow transplant is an option, but limited by the availability of a highly compatible HSC donor. While gene therapy is been explored in several trials, its use is highly limited to developed regions with centers of excellence and well-established healthcare systems (2). Hence, there remains a tremendous unmet medical need to develop alternative treatment strategies for β-thalassemia (3). Occurrence of aberrant splicing is one of the processes that affects β-globin synthesis in β-thalassemia. The (C>G) IVS-2-745 is a splicing mutation within intron 2 of the β-globin gene. It leads to an aberrantly spliced mRNA that incorporates an intron fragment. This results in an in-frame premature termination codon that inhibits β-globin production. Here, we propose the use of uniform 2'-O-methoxyethyl (2'-MOE) splice switching oligos (SSOs) to reverse this aberrant splicing in the pre-mRNA. With these lead SSOs we show aberrant to wild type splice switching. This switching leads to an increase of adult hemoglobin (HbA) up to 80% in erythroid cells from patients with the IVS-2-745 mutation. Furthermore, we demonstrate a restoration of the balance between β-like- and α-globin chains, and up to an 87% reduction in toxic α-heme aggregates. While examining the potential benefit of 2'-MOE-SSOs in a mixed sickle-thalassemic phenotypic setting, we found reduced HbS synthesis and sickle cell formation due to HbA induction. In summary, 2'-MOE-SSOs are a promising therapy for forms of β-thalassemia caused by mutations leading to aberrant splicing.

Background: More than five decades ago, thalassemia major (TDT) was fatal in the first decade of life. Survival and quality of life have improved progressively thanks to the implementation of a significant advance in diagnostic and therapeutic methods, consisting mainly of a frequent transfusion program combined with intensive chelation therapy. Improvement also includes imaging methods used to measure liver and cardiac iron overload. Improved survival has led to a growing number of adults requiring specialised care and counselling for specific life events, such as sexual maturity and acquisition of a family. Aims of the study: The main aim is to present the results of a survey on the marital and paternity status in a large population of adult males with TDT and NTDT living in countries with a high prevalence of thalassemia and a review of current literature using a systematic search for published studies. Results: Ten out of 16 Thalassemia Centres (62.5%) of the ICET-A Network, treating a total of 966 male patients, aged above 18 years with β- thalassemias (738 TDT and 228 NTDT), participated in the study. Of the 966 patients, 240 (24.8%) were married or lived with partners, and 726 (75.2%) unmarried. The mean age at marriage was 29.7 ± 0.3 years. Of 240 patients, 184 (76.6%) had children within the first two years of marriage (2.1 ± 0.1 years, median 2 years, range 1.8 - 2.3 years). The average number of children was 1.32 ± 0.06 (1.27 ± 0.07 in TDT patients and 1.47 ± 0.15 in NTDT patients; p: >0.05). Whatever the modality of conception, 184 patients (76.6%) had one or two children and 1 NTDT patient had 6 children. Nine (4.8%) births were twins. Of 184 patients, 150 (81.5%) had natural conception, 23 (12.5%) required induction of spermatogenesis with gonadotropins (hCG and hMG), 8 (4.3%) needed intracytoplasmic sperm injection (ICSI) and 3 adopted a child. 39 patients with TDT and NTDT asked for medical help as they were unable to father naturally: 7 TDT patients (17.9%) were azoospermic, 17 (37.7%) [13 with TDT and 4 with NTDT] had dysspermia and 15 (33.3%) [13 with TDT and 2 with NTDT] had other “general medical and non-medical conditions”. Conclusions: Our study provides detailed information in a novel area where there are few contemporary data. Understanding the aspects of male reproductive health is important for physicians involved in the care of men with thalassemias to convey the message that prospects for fatherhood are potentially good due to progressive improvements in treatment regimens and supportive care. (www.actabiomedica.it)

We describe the identification of a novel missense mutation in the second zinc finger of KLF1 in two siblings who, based on their genotype, are predicted to suffer from beta thalassemia major but are, in fact, transfusion‐free and in good health. These individuals, as well as two additional members of the same family also carrying this KLF1 mutation, exhibit high levels of fetal hemoglobin (HbF). KLF1 is an erythroid transcription factor, which plays a critical role in the regulation of the developmental switch between fetal and adult hemoglobin by regulating the expression of a multitude of genes including that of BCL11A. The mutation appears to be the main candidate responsible for the beta thalassemia‐ameliorating effect as this segregates with the observed phenotype and also exogenous expression of the KLF1 mutant protein in human erythroid progenitor cells resulted in the induction of γ‐globin, without, however, affecting BCL11A levels. This report adds to the weight of evidence that heterozygous KLF1 mutations can ameliorate the severity of the β‐thalassemia major phenotype.

A review of the literature on COVID-19 pandemic in patients with thalassemias is presented. Globally, the prevalence of COVID-19 among β-thalassemia patients seems to be lower than in general population; associated co-morbidities aggravated the severity of COVID- 19, leading to a poorer prognosis, irrespective of age. A multicenter registry will enhance the understanding of COVID-19 in these patients and will lead to more evidence-based management recommendations. (www.actabiomedica.it)

Objectives: This study aims to investigate, retrospectively, the epidemiological and clinical characteristics, laboratory results, radiologic findings and outcomes of novel coronavirus disease-19 (COVID-19) in patients with transfusion dependent β thalassemia (β-thalassemia major-TM), non-transfusion dependent β thalassemia (β-thalassemia intermedia -TI) and sickle cell disease (SCD). Design, setting: A total of 17 Centers, from 10 countries, following 9,499 patients with hemoglobinopathies participated in the survey. Main outcome measures: Clinical, laboratory and radiologic findings and outcomes of patients with COVID-19 were collected from medical records and summarized. Results: A total of 13 patients, 7 with TM, 3 with TI and 3 with SCD, with confirmed COVID-19, were identified from 6 Centers from different countries. The overall mean age of patients was 33.7±12.3 years (range:13-66); 9/13 (69.2%) patients were females. The commonest symptoms in the 10 symptomatic patients were: fever (80%), cough (70%), headache (60%), fatigue (60%), gastrointestinal symptoms (diarrhea /vomiting/abdominal pain; 50%), tachypnea/dyspnea (40%), and sore throat (40%). Six patients had pneumonia (unilateral, bilateral or multiple opacity) and 4 needed oxygen therapy. An oxygen saturation ≤ 93% was documented in 3 patients at diagnosis. 6/10 patients had an exacerbation of anemia (2 with SCD, associated with back and chest pain in 1 patient), and 3 (

Transfusion-dependent haemoglobinopathies require lifelong iron chelation therapy with one of the three iron chelators (deferiprone, deferasirox, or deferoxamine). Deferasirox and deferiprone are the only two oral chelators used in adult patients with transfusion-dependent haemoglobinopathies. To our knowledge, there are no randomised clinical trials comparing deferiprone, a less expensive iron chelator, with deferasirox in paediatric patients. We aimed to show the non-inferiority of deferiprone versus deferasirox.DEEP-2 was a phase 3, multicentre, randomised trial in paediatric patients (aged 1 month to 18 years) with transfusion-dependent haemoglobinopathies. The study was done in 21 research hospitals and universities in Italy, Egypt, Greece, Albania, Cyprus, Tunisia, and the UK. Participants were receiving at least 150 mL/kg per year of red blood cells for the past 2 years at the time of enrolment, and were receiving deferoxamine (100 mg/kg per day) or deferasirox (40 mg/kg per day; deferasirox is not registered for use in children aged2 years so only deferoxamine was being used in these patients). Any previous chelation treatment was permitted with a 7-day washout period. Patients were randomly assigned 1:1 to receive orally administered daily deferiprone (75-100 mg/kg per day) or daily deferasirox (20-40 mg/kg per day) administered as dispersible tablets, both with dose adjustment for 12 months, stratified by age (10 years and ≥10 years) and balanced by country. The primary efficacy endpoint was based on predefined success criteria for changes in serum ferritin concentration (all patients) and cardiac MRI T2-star (T2*; patients aged10 years) to show non-inferiority of deferiprone versus deferasirox in the per-protocol population, defined as all randomly assigned patients who received the study drugs and had available data for both variables at baseline and after 1 year of treatment, without major protocol violations. Non-inferiority was based on the two-sided 95% CI of the difference in the proportion of patients with treatment success between the two groups and was shown if the lower limit of the two-sided 95% CI was greater than -12·5%. Safety was assessed in all patients who received at least one dose of study drug. This study is registered with EudraCT, 2012-000353-31, and ClinicalTrials.gov, NCT01825512.435 patients were enrolled between March 17, 2014, and June 16, 2016, 393 of whom were randomly assigned to a treatment group (194 to the deferiprone group; 199 to the deferasirox group). 352 (90%) of 390 patients had β-thalassaemia major, 27 (7%) had sickle cell disease, five (1%) had thalassodrepanocytosis, and six (2%) had other haemoglobinopathies. Median follow-up was 379 days (IQR 294-392) for deferiprone and 381 days (350-392) for deferasirox. Non-inferiority of deferiprone versus deferasirox was established (treatment success in 69 [55·2%] of 125 patients assigned deferiprone with primary composite efficacy endpoint data available at baseline and 1 year vs 80 [54·8%] of 146 assigned deferasirox, difference 0·4%; 95% CI -11·9 to 12·6). No significant difference between the groups was shown in the occurrence of serious and drug-related adverse events. Three (2%) cases of reversible agranulocytosis occurred in the 193 patients in the safety analysis in the deferiprone group and two (1%) cases of reversible renal and urinary disorders (one case of each) occurred in the 197 patients in the deferasirox group. Compliance was similar between treatment groups: 183 (95%) of 193 patients in the deferiprone group versus 192 (97%) of 197 patients in the deferisirox group.In paediatric patients with transfusion-dependent haemoglobinopathies, deferiprone was effective and safe in inducing control of iron overload during 12 months of treatment. Considering the need for availability of more chelation treatments in paediatric populations, deferiprone offers a valuable treatment option for this age group.EU Seventh Framework Programme.

In adult thalassemia major (TM) patients, a number of occult and emerging endocrine complications, such as: central hypothyroidism (CH), thyroid cancer, latent hypocortisolism, and growth hormone deficiency (GHD) have emerged and been reported. As the early detection of these complications is essential for appropriate treatment and follow-up, the International Network of Clinicians for Endocrinopathies in Thalassemia and Adolescent Medicine (ICET-A) promoted a survey on these complications in adult TM patients, among physicians (pediatricians, hematologists and endocrinologists) caring for TM patients in different countries. The data reported by 15 countries are presented. The commonest endocrine complications registered in 3.114 TM adults are CH and GHD (4.6 % and 3.0 %, respectively), followed by latent hypocortisolism (1.2%). In 13 patients (0.41%) a cytological papillary or follicular thyroid carcinoma was diagnosed in 11 and 2 patients, respectively, and a lobectomy or thyroidectomy was carried out. Of 202 TM patients below the age of 18 years, the reported endocrine complications were: GHD in 4.5%, latent hypocortisolism in 4.4% and central hypothyrodisim in 0.5%. Transition phase was an area of interest for many clinicians, especially as patients with complex chronic health conditions are responding to new treatments extending their lifespan beyond imagination.. In conclusion, our survey provides a better understanding of physicians’ current clinical practices and beliefs in the detection, prevention and treatment of some endocrine complications prevailing in adult TM patients. Regular surveillance, early diagnosis, treatment and follow-up in a multi-disciplinary specialized setting are recommended. (www.actabiomedica.it)

PV, NR and MMP contributed equally Introduction Patients with red blood cell disorders (RBCD), chronic life threating multisystemic disorders in their severe forms, are likely to be at increased risk of complications from SARS-Cov-2 (Covid-19), but evidence in this population is scarce due to its low frequency and heterogeneous distribution. ERN-EuroBloodNet, the European Reference Network in rare hematological disorders, established a European registry to determine the impact of COVID-19 on RBCD patients and identify risk factors predicting severe outcomes. Methods The ERN-EuroBloodNet registry was established in March 2020 by Vall d'Hebron Research Institute based on REDcap software in accordance with the Regulation (EU) 2016/679 on personal data. The local Research Ethics Committee confirmed that the exceptional case of the pandemic justifies the waiver of informed consent. The ERN-EuroBloodNet registry on RBCD and COVID-19 is endorsed by the European Hematology Association (EHA). Eligible patients had confirmed RBCD and COVID-19. Data collected included demographics, diagnosis, comorbidities, treatments, and COVID-19 (severity grade, clinical manifestations, acute events, treatments, hospitalization, intensive care unit, death). For analysis of COVID-19 severity, two groups were established 1) Mild: asymptomatic or mild symptoms without clinical pneumonia and 2) Severe: pneumonia requiring oxygen/respiratory support and/or admission to intensive care unit. Continuous variables were compared using the Wilcoxon rank-sum test or Kruskall Wallis test, while categorical variables were analyzed using the Chi-square test or Fisher's Exact test. Relevant factors influencing disease or severity were examined by the logistic regression adjusted for age. Results As of June 2021, 42 medical centers from 10 EU countries had registered 373 patients: 191 Sickle cell disease (SCD), 156 Thalassemia major and intermedia (THAL) and 26 other RBCD. 84% of the SCD patients were reported by Spain, Belgium, Italy and The Netherlands and 92% of the THAL patients by Italy and Greece. The mean age of SCD was lower (22.5y) than of THAL (39.6y) with pediatric population accounting for 50.5% in SCD and 9% in THAL (p Age and BMI correlated with COVID-19 severity, as described in the general population (p=0.002, p Potential risk factors such as elevated ferritin, current chelation or history of splenectomy did not confer additional risk for developing severe COVID-19 in any patient group. Only diabetes as a comorbidity correlated with severity grade in SCD (p=0.011) and hypertension in THAL (p=0.014). While severe COVID-19 infection in SCD was associated with both ACS (p Overall, 14.8% RBC patients needed oxygen/respiratory support, 4.4% were admitted to ICU with an overall mortality rate of 0.8% (no deaths were registered in pediatric age), much lower than reported in other similar cohorts. Discussion Results obtained so far show that severe COVID-19 occurs at younger ages in more aggressive forms of SCD and THAL. Current preventive approaches (shielding, vaccinations) focus on age over disease severity. Our data highlights the risk of severe COVID-19 infection in some young patients, particularly those with SS/SB0 SCD, suggesting that immunization should be considered in this pediatric group as well. Results between similar sized cohorts of RBCD patients vary between each other and those presented here, highlighting the importance of collecting all of these small cohorts together to ensure adequate statistical power so that definitive risk factors (eg. age, genotype, comorbidities) can be reliably identified and used to guide management of patients with these rare disorders in the light of the ongoing pandemic. Figure 1 Figure 1. Disclosures Longo: Bristol Myers Squibb: Honoraria; BlueBird Bio: Honoraria. Bardón-Cancho: Novartis Oncology Spain: Research Funding. Flevari: PROTAGONIST COMPANY: Research Funding; ADDMEDICA: Consultancy, Research Funding; BMS: Research Funding; IMARA COMPANY: Research Funding; NOVARTIS COMPANY: Research Funding. Voskaridou: BMS: Consultancy, Research Funding; IMARA: Research Funding; NOVARTIS: Research Funding; ADDMEDICA: Consultancy, Research Funding; GENESIS: Consultancy, Research Funding; PROTAGONIST: Research Funding. Biemond: GBT: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Novo Nordisk: Honoraria; Celgene: Honoraria; Sanquin: Research Funding. Nur: Celgene: Speakers Bureau; Roche: Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Beneitez-Pastor: Agios: Honoraria; Alexion: Honoraria; Novartis: Honoraria; Forma Therapeutics: Honoraria. Pepe: Chiesi Farmaceutici S.p.A: Other: no profit support; Bayer S.p.A.: Other: no profit support. de Montalembert: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Addmedica: Membership on an entity's Board of Directors or advisory committees; BlueBirdBio: Membership on an entity's Board of Directors or advisory committees; Vertex: Membership on an entity's Board of Directors or advisory committees. Glenthøj: Agios: Consultancy; Novo Nordisk: Honoraria; Novartis: Consultancy; Alexion: Research Funding; Bluebird Bio: Consultancy; Bristol Myers Squibb: Consultancy; Saniona: Research Funding; Sanofi: Research Funding. Benghiat: Novartis: Consultancy; BMS: Consultancy. Labarque: Novartis: Consultancy; Bayer: Consultancy; Sobi: Consultancy; NovoNordisk: Consultancy; Octapharma: Consultancy. Diamantidis: Genesis Pharma: Honoraria; Uni-Pharma: Honoraria; Bristol Myers Squibb: Consultancy; IONIS Pharmaceuticals: Research Funding; NOVARTIS, Genesis Pharma SA: Research Funding. Kerkhoffs: Sanofi: Research Funding; Terumo BCT: Research Funding. Iolascon: Celgene: Other: Advisory Board; Bluebird Bio: Other: Advisory Board. Taher: Vifor Pharma: Consultancy, Research Funding; Agios Pharmaceuticals: Consultancy; Ionis Pharmaceuticals: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Colombatti: Novartis: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novonordisk: Membership on an entity's Board of Directors or advisory committees; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees; Addmedica: Membership on an entity's Board of Directors or advisory committees; BlueBirdBio: Research Funding. Mañú Pereira: Novartis: Research Funding; Agios Pharmaceuticals: Research Funding.

The common IVSI-110 (G&gt, A) &beta, thalassemia mutation is a paradigm for intronic disease-causing mutations and their functional repair by non-homologous end joining-mediated disruption. Such mutation-specific repair by disruption of aberrant regulatory elements (DARE) is highly efficient, but to date, no systematic analysis has been performed to evaluate disease-causing mutations as therapeutic targets. Here, DARE was performed in highly characterized erythroid IVSI-110(G&gt, A) transgenic cells and the disruption events were compared with published observations in primary CD34+ cells. DARE achieved the functional correction of &beta, globin expression equally through the removal of causative mutations and through the removal of context sequences, with disruption events and the restriction of indel events close to the cut site closely resembling those seen in primary cells. Correlation of DNA-, RNA-, and protein-level findings then allowed the extrapolation of findings to other mutations by in silico analyses for potential repair based on the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9, Cas12a, and transcription activator-like effector nuclease (TALEN) platforms. The high efficiency of DARE and unexpected freedom of target design render the approach potentially suitable for 14 known thalassemia mutations besides IVSI-110(G&gt, A) and put it forward for several prominent mutations causing other inherited diseases. The application of DARE, therefore, has a wide scope for sustainable personalized advanced therapy medicinal product development for thalassemia and beyond.

Data on the prevalence and type of endocrine disorders in β-thalassemia intermedia (β-TI) patients are scarce. This multicenter study was designed to determine the prevalence of endocrine complications and the associated risk factors in a large group of β-TI patients. In this cross-sectional multicenter study, 726 β-TI patients, aged 2.5–80 years, registered at 12 thalassemic centers, from nine countries, were enrolled during 2017. In a subgroup of 522 patients (mean age 30.8 ± 12.1; range: 2.5–80 years) from Qatar, Iran, Oman, Cyprus, and Jordan detailed data were available. Overall, the most prevalent complications were osteopenia/osteoporosis (22.3%), hypogonadism (10.1%), and primary hypothyroidism (5.3%). In the subgroup multivariate analysis, older age was a risk factor for osteoporosis (Odds ratio: 7.870, 95% CI: 4.729–13.099, P

Background: Classification of phenotype severity in patients with beta-thalassaemia has so far relied mainly on expert opinion using parameters of genotype, clinical features at diagnosis, and transfusion requirement. The aim of this study was to use a large dataset of patients with beta-thalassaemia and evaluate a classification system based on onset variables agreed on by an international expert group, including age at diagnosis, at first transfusion, and at first iron chelation. Methods: A retrospective dataset of 7910 patients with homozygous or compound heterozygous beta-thalassaemia was used and subjected to cluster and classification analysis starting with the onset variables. Results: Cluster analysis suggested that three clusters with minimal overlapping exist. Three phenotype severity classes (mild, moderate, severe) were accordingly assigned which showed statistically significant descending variation of age at diagnosis and start of transfusion and iron chelation. The estimated classification error rate was only 3.07% with an accuracy of 96.93%. It was evident that severe patients had higher blood requirement and iron overload levels and showed a younger age for mortality especially from heart disease. Although mild and moderate patients showed the opposite in gradual severity, they still showed evidence of high morbidity rate for complications that require longer time to manifest (eg liver damage and hepatocellular carcinoma). Conclusion: Age of diagnosis and start of transfusion and iron chelation distinguish three classes of phenotype severity in beta-thalassaemia that carry unique clinical profiles. Further prospective studies are recommended to validate these findings. Funding Statement: The authors state: "None" Declaration of Interests: The authors state: " None to disclose." Ethics Approval Statement: An International Health Repository (IHR) protocol, approved on May 25th, 2017 by the Italian Ethical Committee (EudraCT and Sponsor's Protocol Code Numbers were 2017-004457-17 and 143AOR2017) was established to allow collection of relevant data.

Red Blood Cells (RBCs) samples from healthy volunteers, β-thalassaemia major and β-thalassaemia intermedia patients were studied by means of 57Fe Mossbauer Spectroscopy (MS). The MS spectra from the RBCs of the thalassaemia patients showed the existence of significant amounts of ferritin-like iron, and in particular in the 82% of the β-thalassaemia intermedia ones. Also, β-thalassaemia intermedia patients were found to have higher Liver Iron Concentration (LIC) when compared to the β-thalassaemia major patients, as assessed by MRI exams, even though they had on an average about half the serum ferritin levels.

Due to the recent alarming increase in the incidence of hepatocellular carcinoma (HCC) in thalassemias, the aim of the present report is to review briefly the frequency, the major risk factors and the surveillance of HCC in β-thalassemias. Over the past 33 years, 153 cases of HCC were reported in patients with thalassemia, mainly in Italy, and Greece. Among HCV-infected patients additional factors promoting development of HCC, included: advanced age, male sex, chronic hepatitis B (CHB) coinfection, and iron overload. For early diagnosis of HCC sequential ultrasound screening is recommended especially for thalassemia patients with chronic hepatitis C (CHC), that coincide with (one or more) additional risk factors for HCC. Here we report also the preliminary data of thalassemic patients, above the age of 30 years, followed in 13 different centers. The total number of enrolled patients was 1,313 (males: 612 and 701 females). The prevalence of HCC in thalassemia major patients [characterized by transfusion-dependency (TDT)] and thalassemia intermedia [characterized by nontransfusion dependency (NTDT)] was 1.68 % and 1.98 % ,respectively The lowest age at diagnosis was 36 years in TDT and 47 years in NTDT patients.We hope that our review can be used to develop more refined and prospective analyses of HCC magnitude and risk in patients with thalassemia, and to define specific international guidelines to support clinicians for an early diagnosis and treatment of HCC in thalassemic patients.

Hypoparathyroidism (HPT) is a rare disease with leading symptoms of hypocalcemia, associated with high serum phosphorus levels and absent or inappropriately low levels of parathyroid hormone (PTH). In patients with thalassemias it is mainly attributed to transfusional iron overload, and suboptimal iron chelation therapy. The main objectives of this survey were to provide data on the prevalence, demographic and clinical features of HPT in thalassemia major (TM) and intermedia (TI) patients living in different countries, and to assess its impact in clinical medical practice. A questionnaire was sent to all Thalassemia Centres participating to the International Network of Clinicians for Endocrinopathies in Thalassemia and Adolescence Medicine (ICET-A) Network. Seventeen centers, treating a total of 3023 TM and 739 TI patients, participated to the study. HPT was reported in 206 (6.8%) TM patients and 33 (4.4%) TI patients. In general, ages ranged from 10.5 to 57 years for the TM group and from 20 to 54 years for the TI group. Of the 206 TM patients and 33 TI patients with HPT, 117 (48.9%) had a serum ferritin level >2.500 ng/ml (54.3% TM and 15.1% TI patients) at the last observation. Hypocalcemia varied in its clinical presentation from an asymptomatic biochemical abnormality to a life-threatening condition, requiring hospitalization. Calcium and vitamin D metabolites are currently the cornerstone of therapy in HPT. In TM patients, HPT was preceded or followed by other endocrine and non-endocrine complications. Growth retardation and hypogonadism were the most common complications (53.3% and 67.4%, respectively). Although endocrine complications were more common in patients with TM, non-transfused or infrequently transfused patients with TI suffered a similar spectrum of complications but at a lower rate than their regularly transfused counterparts. In conclusion, although a large international registry would help to better define the prevalence, comorbidities and best treatment of HPT, through the result of this survey we hope to give a clearer understanding of the burden of this disease and its unmet needs. HPT requires lifelong therapy with vitamin D or metabolites and is often associated with complications and comorbidities. Therefore, it is important for endocrinologists and other physicians, who care for these patients, to be aware of recent advances of this disorder.(www.actabiomedica.it)

The β thalassemia trait is associated with over 300 mutations in the β-globin gene that lead to reduced (β+ allele) or absent (β0 allele) synthesis of the β globin chain. A subset of these mutations affect the canonic splicing of the β globin mRNA. Such mutations activate aberrant splice sites, which lead to an altered splicing pathway and consequently affects protein synthesis. The (C>G) IVS-2-745 mutation is common in South Eastern Europe, Cyprus, Lebanon, India, Malaysia, and Indonesia. This mutation, located within intron 2 of the β-globin gene, creates an aberrant 5' splice site at nucleotide 745 of intron 2 and activates a cryptic 3' splice site within the same intron. Portions of the intronic sequence are incorrectly retained in the spliced mutant mRNA. The mutation results in a premature stop codon that prevents proper mRNA translation and causes a β‐globin deficiency, resulting in β‐thalassemia. The IVS-2-745 allele has the functional splice sites preserved, but produces a significantly reduced level of correctly spliced β-globin mRNA and results in only marginal synthesis of HbA. Therefore, the IVS-2-745 mutation in homozygosity leads to severe transfusion-dependent thalassemia major. Taking advantage of conserved canonical splice sites in defective β‐globin genes, such as IVS-2-745, recently developed approaches show that by targeting the aberrant splice sites it is possible to circumvent the aberrant splice site and restore the normal β-globin splicing pattern. We sought to use uniform 2'-O-methoxyethyl (2'-MOE) splice switching oligos (SSOs) to reverse aberrant splicing in the pre-mRNA for the IVS-2-745 mutation. Using these SSOs, we show effective aberrant-to-wild-type splice switching. This leads to an increase in adult hemoglobin (HbA) by up to 80% in erythroid cells from patients with the IVS-2-745 mutation. Furthermore, we demonstrate a restoration of the balance between β-like- and α-globin chains, and up to an 87% reduction in α-heme aggregates. While examining the potential benefit of 2'-MOE-SSOs in a sickle/IVS-2-745-thalassemic genotype setting, we found that use of these oligos restored production of HbA and reduced HbS synthesis, which ultimately lessened cell sickling under hypoxic conditions. We confirmed increased WT β-globin expression in specimens treated with 2'-MOE-SSOs with semi- and quantitative methods (RT and Q-PCR), and further supported this evidence using a direct quantification method (ddPCR). Compared to treated specimens heterozygous for IVS-2-745 , homozygous specimens showed elevated WT HbA, reflecting the additive effect of targeting the aberrant splicing of both alleles as opposed to a single IVS-2-745 allele. In fact, while 2'-MOE-SSOs significantly reduced aberrant splicing, leading to a consequent 60% increase in HbA levels in specimens from patients with a β0/IVS-2-745 genotype, the same oligos produced a more robust effect in specimens with a homozygous IVS-2-745 genotype, resulting in an 80% increase in HbA levels. This level of increase could potentially be curative for patients with this particular genotype. Moreover, we compared the effect of 2'-MOE-SSOs treatment to a lentiviral vector carrying a WT β-globin gene. In this comparative assay, β0/IVS-2-745 cells treated with 2'-MOE-SSOs or the lentivector (with 1.13 copies integrated per genome) lead to a similar increase in HbA (50%). This suggests that the oligo-based technology is a competitive approach and a viable alternative to gene addition therapy to overcome anemia in IVS-2-745 β-thalassemia. In summary, 2'-MOE-SSOs are promising therapeutic tools for certain forms of β-thalassemia caused by aberrant splicing. Their ability to correct the underlying splicing defect offers a pharmacological treatment that is direct, specific, and accessible. In comparison, gene therapy approaches utilizing gene addition or editing are primarily available in advanced medical care environment resulting in an unfulfilled demand in regions where such conditions are not readily available. The restoration of target gene activity reported here suggests that this treatment strategy could be applicable to other forms of thalassemia resulting from mutations affecting splicing. This could have, with an effective method of delivery, potential clinical utility in helping patients reduce their transfusion dependence or even achieving transfusion independence. Disclosures Dong: Aruvant Sciences INC: Employment. Motta:Sanofi-Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees. Guo:Ionis Pharmaceutical, INC: Employment, Other: shareholders. Peralta:Ionis Pharmaceutical, Inc: Employment. Freier:Ionis Pharmaceuticals: Employment. Watt:Ionis Pharmaceuticals: Employment. Manwani:Novartis: Consultancy; Pfizer: Consultancy; GBT: Consultancy, Research Funding. Cappellini:Genzyme/Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Honoraria; Vifor Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Membership on an entity's Board of Directors or advisory committees. Abdulmalik:The Children's Hospital of Philadelphia: Patents & Royalties: Provisional Patent. Rivella:Meira GTx, Ionis Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Disc medicine, Protagonist, LIPC, Meira GTx: Consultancy.

β-Thalassaemia (β-thal), as an early-onset severe monogenic blood disorder, is an ideal target for advanced therapy medicinal product (ATMP) development based on gene addition or gene correction. A...

In this paper, blood and tissues samples from one normal and one thalassaemic lab-mice were studied using 57Fe Mossbauer spectroscopy at 78K for the first time. In contrast to human patients, these laboratory mice did not receive any medical treatment, thus the iron components present in the samples are not altered. The measured Moessbauer spectra of the blood, liver and spleen samples of the thalassaemic mouse were found to be different in the shape and iron content as compared to the corresponding spectra of the normal mouse. This result demonstrates the further exploitation of the thalassaemic mouse model to study thalassaemia in more details by means of Moessbauer spectroscopy., Comment: 13 pages including 3 tables and 5 figures

The diagnosis of beta thalassemia intermedia (BTI) is mainly based on the severity of clinical phenotype. It is associated with a wide range of specific complications including extramedullary hematopoiesis, leg ulcers, gallstones, hypercoagulable state, pulmonary hypertension (PHT), endocrine disorders and osteoporosis. The commonest endocrine complication in beta thalassemia major (TM) patients is hypogonadism followed by hypothyroidism and diabetes but the data on endocrine disorders in BTI patients are scarce. The aim of this study is to determine the prevalence of endocrine complications in a large series of BTI patients. Methods: In this multi-countries cross-sectional study, all BTI patients registered at 12 thalassemic treatment centers in Iran (2 Centers), Italy (2 Centers), Greece, Turkey (2 centers), Oman, Qatar, Jordan, Cyprus and United Kingdom were enrolled during 2017. Non transfusion- dependent beta-thalassemia patients or those who received blood transfusion 3-4 times or less annually, were evaluated. Required information was collected from medical records using a designed questionnaire. Demographic data, clinical characteristics and laboratory data included age, sex, splenectomy, type of treatment (blood transfusion, iron chelation, hydroxyurea), hormonal assays, bone mineral density, calcium -phosphorous metabolism, serum ferritin, liver function tests, fetal and total hemoglobin levels,, platelet and nucleated red blood cell counts, were collected. Results: A total of 721 BTI patients were enrolled in the survey from 9 countries. The most prevalent disease-related complications were osteoporosis (21.6%) and hypogonadism (12.6%) followed by: central hypothyroidism (8.3%), non-insulin-dependent diabetes mellitus (7.8%), primary hypothyroidism (5.5%), insulin-dependent diabetes mellitus (4.2%), hypoparathyroidism (2.2%), growth hormone deficiency (1.1%), adrenal mass (1%) and thyroid cancer (0.5%). Conclusion: This study evaluated the largest cohort of BTI patients with endocrine disorders. Although BTI patients are non-transfusion dependent or only occasionally transfused, iron-overload due to increased intestinal iron absorption and enhanced bone marrow activity cause endocrine disorders and osteoporosis. This study demonstrate that although endocrine complications are less common in patients with BTI compared to data reported in literature in TM patients , a regular monitoring with timely diagnosis and proper management underscoring on osteoporosis and gonadal disorders are crucial to prevent endocrine complications in these patients. Disclosures No relevant conflicts of interest to declare.

Morbidity and mortality related to thalassemia have been reduced significantly with modern medical treatment, and quality of life (QOL) should now be considered an important index of effective health care. An assessment of QOL differs from other forms of medical assessment in that it focuses on the individuals' own views of their well-being and assesses other aspects of life, giving a more holistic view of well-being. There is very little published work on evaluation of QOL in thalassemia. A suitable tool should be reproducible, sensitive to the major features of the condition that affect patients' lives, and applicable in the range of different cultural, age, and social settings. Such an instrument would be valuable in evaluating new forms of treatment and in comparing health outcomes between different clinics. Two instruments have been assessed, one derived from the WHOQOL-100 questionnaire, and one designed specifically for thalassemia, which assesses psychosocial and clinical burden, as they affect adult patients, parents, and children. Further studies are required to develop and assess such tools for use in thalassemia. Another approach is to seek patients' own views of their routine treatment and the extent to which medical treatment affects QOL. Results from patient questionnaires in the United Kingdom and Cyprus are consistent in finding problems with organization of transfusions, insufficient options with chelation therapy, and poor communication. Practical measures could be taken to address these issues.

Background: Multi-transfused thalassemia major (TM) patients frequently develop severe endocrine complications, mainly due to iron overload, anemia and chronic liver disease, which require prompt diagnosis, treatment and follow-up by specialists. The most common endocrine complication documented is hypogonadotropic hypogonadism which increases with age and associated comorbidities. It is thus important for physicians to have a clear understanding of the pathophysiology and management of this disorder. Also to be aware of the side effects, contraindications and monitoring of sex steroid therapy. In this paper practical ICET-A recommendations for the management of hypogonadism in adult females with TM are addressed. Methods: In March 2015, the Coordinator of the International Network of Clinicians for Endocrinopathies in Thalassemia and Adolescent Medicine (ICET-A) conducted a two-step survey to assess the attitudes and practices of doctors in the ICET-A network taking care of adult female TM patients with hypogonadism. They were clinically characterized by the absence of pubertal development, or discontinuation or regression of the maturation of secondary sex characteristics, and biochemically by persistent low FSH, LH and estradiol levels. Recently a supplementary survey on adult female hypogonadism in TM was undertaken within the ICET-A network. Results: The completed questionnaires were returned by 16 of 27 specialists (59.2%) following 590 female TM patients over the age of 18 years; 315 patients (53.3%) had hypogonadism and only 245 (74.6%) were on hormone replacement therapy (HRT). Contraceptive oral pills (COC) were the first treatment choice in 11 centres (68.7%). A wide range of COCs were used with different progestin contents. In general, the patients’ compliance to treatment was reported as good in 81.2 % of centres. The frequency of required tests for follow-up HRT, in addition to the regular check-up for thalassemia, was variable in the participating centres. Conclusions: Doctors taking care of TM patients should have sound knowledge of the pathophysiology of hypogonadism in adult females with TM. They should know the potential effects of HRT including advantages and disadvantages of estrogen and progestins. Moreover, they should keep in consideration the emotional needs of these patients dreaming to attain a full pubertal development. Key words: Thalassemia, hypogonadism, hormone replacement therapy, benefits and disadvantages

Hemoglobinopathies are the most common inherited blood disorders. World Health Organization statistics show that in the Mediterranean, Eastern European, and Middle Eastern regions, frequencies range from 0.1 to 4.9/1000 of live births. The mutation known as IVS2-745 is relatively common in the regions of Spain, Jordan, Romania, and Serbia (Ithanet Database, http://www.ithanet.eu/db/ithamaps), reaching as high as 15-20% of beta-thalassemia mutations in these regions. The IVS2-745 is a splicing mutation that occurs in intron 2 of the beta-globin gene and results in an aberrantly spliced mRNA that incorporates an extra exon and premature stop codon. Here we report novel uniform 2'-O-methoxyethyl (2'-MOE) splice switching oligos (SSOs) that reverse the aberrant splicing and restore up to 80% adult hemoglobin (HbA) production in vitro. Uniform 2'-MOE SSOs do not mediate RNase H degradation when they bind their targets; therefore, they can be used to redirect the splicing machinery and restore WT splicing. After generating mouse erythroleukemia cells that carry the human IVS2-745 mutated beta-globin gene, lead 2'-MOE SSOs targeting the 745 mRNA were raised against these cell lines. With these lead SSOs we have demonstrated aberrant 745 to WT splice switching in 5 patient samples. CD34+ cells were isolated from the blood of four 745/β0 compound heterozygotes and one 745/ 745 homozygote (Breda et al, PloS One 2012). After CD34+ expansion, cells were differentiated to the red cell lineage and treated via syringe loading or lipofectamine transfection with 2'-MOE SSOs. Up to 80% HbA protein production was restored with 2'MOE-SSO treatment in the 745 homozygote patient sample (Figure 1), and up to 60% HbA in multiple 745 compound heterozygote specimens. Compared to 3-6% HbA in scramble treated controls, this represents up to a 20-fold increase in HbA with treatment. In addition to HbA production, we have shown improvement in other parameters characteristic in beta-thalassemia, such as the imbalance of alpha and beta chains and the accumulation of toxic alpha-only homotetramers. 2'MOE SSOs are able to reinstate balance of beta- to alpha-like chains, which resulted in a near elimination of toxic alpha-only homotetramers in the homozygote cell lysate as detected by HPLC (Figure 1). We further proved the benefit of 2'MOE SSOs in a 745/Sickle model system, where in vitro sickling was significantly reduced as a result of increased levels of HbA. To create this model system, we transduced CD34+ cells from a homozygous sickle patient specimen with a lentivirus expressing human IVS2-745 beta-globin. With vector copy numbers ~2, this system replicates what a single allele would do, as the 2 endogenous sickle alleles are equally matched. Upon differentiation and exposure to hypoxia, in vitro sickling was reduced by 50% in 2'MOE-SSO treated samples as compared to scramble controls (Figure 2). In summary, 2'MOE-SSOs are a promising therapy for certain splicing forms of beta-thalassemia. Their ability to correct the underlying splicing cause offers a pharmacological treatment that is both direct and specific. As such, this therapy could help patients reduce their transfusion dependence or even reach transfusion independence. Disclosures Guo: Ionis Pharmaceuticals: Employment, Equity Ownership. Peralta:Ionis Pharmacueticals: Employment. Cappellini:Celgene: Membership on an entity's Board of Directors or advisory committees; Genzyme-Sanofi: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Apolipoprotein E (APOE) plays an important role in the multifactorial etiology of both cardiovascular disease and Alzheimer's disease. Polymerase chain reaction (PCR) was used to investigate the APOE gene polymorphism in 335 unrelated Greek Cypriots living on the island of Cyprus. For the most common APOE genotypes, the Greek Cypriots followed the general Caucasian European pattern of having higher genotypic frequencies of E3/3, followed by E3/4, and then E2/3. Among the European populations compared, Greek Cypriots exhibited the lowest relative frequency of the E3/4 genotype (12.83%). Also, the relative frequencies of the E2 and E4 alleles in Greek Cypriots were among the lowest around the world (5.4% and 7.0%, respectively). This was also demonstrated by using the complete and the average clustering methods of analysis where the APOE allele relative frequencies in Greek Cypriots were compared to 46 other populations. The Greek Cypriot population in these analyses clustered with populations mainly from south Europe and Japan which have low E2 and E4 allele frequencies. The Greek Cypriot population will be studied further for elucidating the effect(s) and the role of APOE in cardiovascular disease and the APOE4 allele as a possible metabolic factor affecting the rate of expression of both Alzheimer's disease and vascular dementia.

A large number of patients with thalassemia major have been born and treated exclusively in Cyprus. They have been managed according to standard international practice, but few have been transplanted. In 1999, a combination chelation regime with desferrioxamine and deferiprone was introduced. We analyzed survival trends in Cypriots and tried to identify factors associated with prolonged survival.We had incomplete information on births pre-1974 and complete information from 1974 onwards. Clinical data were incomplete pre-1980 and complete thereafter. We analyzed data on 539 patients born after 1960 and followed over the period 1980 to the end of 2004.There were 58 deaths, 31 (53.4%) of which where due to cardiac causes. In the complete birth cohort of 284 patients born after 1974, survival (95% CI) at 10, 20 and 30 years was 100% (0); 98.5% (96.1-99.4) and 92.7% (86.7-96.1) respectively. There was a significant trend of increasing cardiac deaths between 1980 and 2000 (p0.001) and a decline after 2000 (p=0.06). In multivariate survival analysis, protective effects were found for female sex (hazard ratio, 0.37, 95% CI 0.21-0.66; p0.001), and post-2000 follow-up (hazard ratio, 0.44, 95% CI 0.20-0.99; p0.05), but not for genotype, treatment center or birth cohort.Most patients born after 1974 survive to at least the age of 30. There has been a marked improvement in survival for patients of all ages since 2000, which may be due to the introduction of combination chelation therapy.

Effective and convenient iron chelation remains one of the main targets of clinical management of thalassemia major. The combined treatment with desferrioxamine and deferiprone could have an increased chelation efficacy and sometimes allow drug doses and toxicity to be reduced and the number of days of desferrioxamine infusion to be decreased, improving compliance and quality of life.We used combined therapy with desferrioxamine and deferiprone to treat 79 patients with severe iron overload (serum ferritin higher than 3000 ng/mL) who had low compliance with subcutaneous desferrioxamine.Total therapy exposure was 201 patient-years. Three patients developed agranulocytosis and seven mild neutropenia. Other adverse effects were nausea, vomiting, abdominal pain, increased concentrations of liver transaminases and joint pain. The efficacy of combined therapy was evaluated in 64 patients treated for at least 12 months. Ferritin decreased from 5243+/-2345 to 3439+/-2446 ng/mL, p0.001). Mean urinary iron excretion during combined therapy was double that with desferrioxamine or deferiprone monotherapy. In 20 patients receiving heart therapy at baseline, left ventricular ejection fraction increased from 48.6+/-9% to 57+/-6% (p=0.0001) over 12 to 57 months, without modifying the cardiac treatment.Continuous deferiprone treatment with intermittent administration of subcutaneous desferrioxamine is a practical and effective procedure to decrease severe iron overload in patients with thalassemia major. This study also shows that the combined therapy is associated with an improvement in heart function.

Aim: In humans, fetal hemoglobin (HbF) production is controlled by many intricate mechanisms that, to date, remain only partly understood. Patients & methods: Pharmacogenomic analysis of the effects of hydroxyurea (HU) on HbF production was undertaken in a collection of Hellenic βthalassemia and sickle cell disease (SCD) compound heterozygotes and a collection of healthy and KLF1-haploinsufficient Maltese adults, to identify genomic signatures that follow high HbF patterns. Results: KLF10 emerged as a top candidate. Moreover, genotype analysis of βthalassemia major and intermedia patients and an independent cohort of βthalassemia/SCD compound heterozygous patients that do or do not respond to HU treatment showed that the homozygous mutant state of a tagSNP in the KLF10 3’UTR is not present in βthalassemia intermedia patients and is underrepresented in βthalassemia/SCD compound heterozygous patients that respond well to HU treatment. Conclusion: These data suggest that KLF10 may constitute a pharmacogenomic marker to discriminate between response and nonresponse to HU treatment. Original submitted: 2 May 2012; Revision submitted: 17 July 2012

Haemoglobinopathies are the most common monogenic diseases, posing a major public health challenge worldwide. Cyprus has one the highest prevalences of thalassaemia in the world and has been the first country to introduce a successful population-wide prevention programme, based on premarital screening. In this study, we report the most significant and comprehensive update on the status of haemoglobinopathies in Cyprus for at least two decades. First, we identified and analysed all known 592 β-thalassaemia patients and 595 Hb H disease patients in Cyprus. Moreover, we report the molecular spectrum of α-, β- and δ-globin gene mutations in the population and their geographic distribution, using a set of 13824 carriers genotyped from 1995 to 2015 and estimate relative allele frequencies in carriers of β- and δ-globin gene mutations. Notably, several mutations are reported for the first time in the Cypriot population, whereas important differences are observed in the distribution of mutations across different districts of the island.