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IntroductionPyruvate kinase (PK) deficiency is a rare, under-recognised, hereditary condition that leads to chronic haemolytic anaemia and potentially serious secondary complications, such as iron overload, cholecystitis, pulmonary hypertension and extramedullary haematopoiesis. It is an autosomal recessive disease caused by homozygous or compound heterozygous mutations in thePKLRgene. Due to its rarity and clinical heterogeneity, information on the natural history and long-term clinical course of PK deficiency is limited, presenting major challenges to patient management, the development of new therapies and establishing disease-specific treatment recommendations. The Pyruvate Kinase Deficiency Global Longitudinal (Peak) Registry is an initiative to address the gaps in the knowledge of PK deficiency. This manuscript describes the objectives, study design and methodology for the Peak Registry.Methods and analysisThe Peak Registry is an observational, longitudinal, global registry of adult and paediatric patients with a genetically confirmed diagnosis of PK deficiency. The Peak Steering Committee is composed of 11 clinicians and researchers with experience in the diagnosis and management of PK deficiency from 10 countries, a patient representative and representatives from the sponsor (Agios Pharmaceuticals). The registry objective is to foster an understanding of the longitudinal clinical implications of PK deficiency, including its natural history, treatments and outcomes, and variability in clinical care. The aim is to enrol up to 500 participants from approximately 60 study centres across 20 countries over 7 years, with between 2 and 9 years of follow-up. Data will include demographics, diagnosis history, genotyping, transfusion history, relevant clinical events, medications, emergency room visits and hospitalisations.Ethics and disseminationRegistry protocol and informed consent forms are approved by institutional review boards/independent ethics committees at each study site. The study is being conducted in accordance with the Declaration of Helsinki. Registry data will be published in peer-reviewed journal articles and conference publications.Trial registration numberNCT03481738.

Sickle cell disease (SCD) and thalassemias are the most common monogenic diseases in the world. The number of migrants and refugees in Europe and Turkey, in the past decade, has increased dramatically due to war, violence or prosecutions in their homeland. Prevention and management of haemoglobin disorders is well established and managed in countries where these conditions were traditionally endemic or in countries that have a longstanding tradition of receiving migrants. Therefore, preventive and diagnostic programmes regarding hemoglobinopathies in immigrant populations have been implemented. The purpose of this paper it to report a summary of the experience gained in Italy, Spain and Turkey in migrants, asylum seekers and refugees. (www.actabiomedica.it)

INTRODUCTION NlmCategory: BACKGROUND content: In the current mobility and globalization context, there is a growing need to identify potential changes on the pattern of diseases in the European Union (EU)/European Economic Area (EEA) and provide accurate diagnosis and treatment for the population. The pattern of rare communicable diseases that can affect people returning to EU/EEA from travel abroad, visiting EU/EEA or establishing in the EU/EEA is of special relevance. The objective of this manuscript is to give an overview about the EURaDMoG study and discuss the feasibility of establishing a European network on rare communicable diseases and other rare conditions linked to mobility and globalization. - Label: METHODS NlmCategory: METHODS content: We undertook a three-steps process where we first conducted a narrative review to estimate the prevalence and incidence and to list rare communicable and non-communicable diseases linked to mobility and globalization in the EU/EEA; second, we organized an international consultation workshop with experts in the diseases previously selected; and finally, the feasibility study analysed how successful a European expert network on rare diseases linked to mobility and globalization focused on health care provision would be, accounting for different operational and also sustainability criteria. - Label: RESULTS NlmCategory: RESULTS content: "First, considering the areas or topics that the network should cover, it was concluded that communicable and non-communicable rare diseases linked to mobility and globalization should be differentiated. Second, since all non-communicable rare diseases linked to mobility and globalization identified are already covered by different European Reference Networks (ERNs), there is no need for them to be included in a new European network. Three scenarios were considered for establishing a potential European network for rare communicable diseases linked to Mobility and Globalisation with a focus on Health Care provision: 1) To maintain the current situation \"Status Quo\" scenario; 2) to create a specific European expert network (EEN) on rare communicable diseases linked to mobility and globalisation; 3) to develop a new ERN on communicable rare diseases linked to mobility and globalisation." - Label: CONCLUSIONS NlmCategory: CONCLUSIONS content: Since the focus is the provision of health care, an ERN could have the potential to better boost the quality of care being facilitated by technological tools and online platforms that permit the safe and ethically acceptable exchange of data. However, this potential new network should not eclipse current existing networks and they should be complementary.

COVID-19 is an infectious disease caused by the SARS-CoV-2 virus. Most people with COVID-19 have a mild to moderate respiratory illness; others experience severe illness, such as COVID-19 pneumonia. The first and most accessible diagnostic information is from symptoms and signs from clinical examination. Infected patients present with a variety of manifestations. Formal diagnosis requires laboratory analysis of nose and throat samples, or imaging tests like CT scans. Emerging data suggest that coronavirus disease 2019 (COVID-19) has extrapulmonary manifestations. Sometimes these extra-respiratory manifestations may be the initial or only symptom of COVID-19, prior to fever or respiratory manifestations. In summary, our concise review shows that there is a wide range of symptoms that can be presented by COVID-19 patients. Extra-respiratory manifestations of SARS-CoV-2 infection have recently been observed in the rapidly increasing number of COVID-19 cases. Considering the broad spectrum of clinical manifestations and the increasing worldwide burden of the disease, there is an urgent need to rapidly scale up the diagnostic capacity to detect COVID-19 and its complications. (www.actabiomedica.it)

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 (

Hereditary red blood cell (RBC) membranopathies are characterized by mutations in genes encoding skeletal proteins that alter the membrane complex structure. Hereditary spherocytosis (HS) is the most common inherited RBC membranopathy leading to hereditary hemolytic anemia with a worldwide distribution and an estimated prevalence, in Europe, of about 1:2000 individuals. The recent availability of targeted next generation sequencing (t-NGS) and its combination with RBC deformability measured with a laser-assisted optical rotational ektacytometer (LoRRca) has demonstrated to be the most powerful contribution to lower the percentage of hereditary hemolytic anemia undiagnosed cases. In order to know the kind and frequency of RBC membrane mutations in our geographical area (Catalonia) and to better understand their pathophysiology, 42 unrelated, non-transfusion-dependent (NTD) patients with hereditary hemolytic anemia have been studied by combining t-NGS and LoRRca. The osmoscan module of LoRRca provides three rheological profiles that reflect the maximal deformability (EImax), osmotic fragility (Omin), and hydration state (Ohyper) of RBCs and contribute to a better understanding of the contribution RBC rheology to the severity of anemia. From the 42 patients studied, 37 were suspected to be a RBC membrane defect due to phenotypic characteristics and abnormal RBC morphology and, from these, in 31 patients (83.8% of cases) the mutation was identified by t-NGS. No definite diagnosis was achieved in 11 patients (26.2% of cases), including 6 out of 37 cases, with suspected membranopathy, and 5 with unclassifiable HHA. In all these undiagnosed patients, the existence of hemoglobinopathy and/or enzymopathy was ruled out by conventional methods.

For rare haematological diseases (RHD), the first question to be answered is if patients with benign red blood cell (RBC) defects like haemoglobinopathies, membranopathies and enzymopathies are more vulnerable to COVID-19 infection. Up to now, there is no yet literature on the subject, but, like in general population, the presence of comorbidities such as diabetes, heart disease, pulmonary hypertension, reduced kidney and/or liver function, worsen the effects of the infection. Splenectomy may be an additional risk factor. (www.actabiomedica.it)

Here we describe a retrospective study of a 10-year-old girl, adopted from India, and referred to the Rare Anemias Unit for the diagnosis of a severe haemolytic anaemia of unknown etiology. Blood film examination revealed markedly abnormal red cell morphology characterised by a mixture of very pale (hypochromic) cells with basophilic stippling and macrocytic cells containing coarse basophilic dots and an amorphous material of unknown origin. With a presumptive diagnosis of pyruvate kinase deficiency (PK), the patient had been splenectomised at 7 years of age with a partial recovery of the anaemia and a decrease of the blood transfusion rate. Three years after splenectomy, the patient was revisited and a haemoglobin stability test was performed with a positive result. Accordingly, the correct diagnosis was an unstable haemoglobinopathy. Targeted next generation sequencing (t-NGS) revealed haemoglobin Bristol-Alesha, a hyper unstable haemoglobinopathy associated with severe haemolytic anaemia. Since unstable haemoglobins do not necessarily have specific red cell morphological abnormalities, our findings reinforce the need to include, the haemoglobin stability test, in the first diagnostic approach of hemolytic anaemias of unknown etiology.

Background: Pyruvate kinase (PK) deficiency is a rare, congenital, glycolytic enzyme defect that leads to chronic hemolytic anemia. Despite existing supportive therapies, many patients (pts) experience significant complications, including iron overload (FeO). FeO in PK deficiency is clinically underappreciated and may cause long-term organ damage and impact growth in children and adults. Objective: To describe characteristics and disease burden of pts with PK deficiency and a history of FeO by age group (pediatric cohort, Methods: The Peak Registry is an ongoing, global retrospective and prospective observational study of adult and pediatric pts diagnosed with PK deficiency. For this analysis, pts were considered to have a history of FeO if they had ever received 1) chelation therapy; or 2) phlebotomy for removal of iron; or within 3 months of enrollment had 3) ferritin >1000 ng/mL; or 4) liver MRI >3 mg Fe/g dry weight; or 5) FerriScan® >3 mg Fe/g dry weight; or 6) cardiac T2* MRI ≤20 ms. Data on demographics, medical history, treatment, clinical assessment, and laboratory testing collected at study enrollment were summarized descriptively among pts with a history of FeO. Results: As of the 24Mar2020 data cut, 57/140 (40.7%) enrolled pts had a history of FeO (adult cohort N=28; pediatric cohort N=29); data from these 57 pts were used for this analysis. Average age at enrollment for adult and pediatric cohorts was 37.3 yrs (standard deviation [SD]: 14.80) and 7.7 yrs (SD: 4.64), respectively. PKLR genotype information was available for 21 adult and 15 pediatric pts; among the 21 adults, 13 (61.9%) had two missense mutations (M/M), 7 (33.3%) had one missense/one non-missense mutation (M/NM), and 1 (4.8%) had two non-missense mutations (NM/NM); among the 15 pediatric pts, 3 (20%) had M/M, 7 (46.7%) had M/NM, and 5 (33.3%) had NM/NM (Table). Among pts with known transfusion status, 6/56 FeO pts (10.7%) had never received a transfusion, with all other pts having received ≥1 prior transfusion (ever-transfused). Median hemoglobin (Hb) level at the time of enrollment was 8.6 g/dL (range: 6.7-12.5) in ever-transfused adults and 10.9 g/dL (9.3-11.3) in never-transfused adults. In pediatric pts (all ever-transfused), median Hb at enrollment was 8.3 g/dL (6.8-10.8). Most ever-transfused pts had received chelation therapy (18/20 [90.0%] adults, 28/29 [96.6%] pediatrics). Of the 6 pts who had never been transfused, 3 (50.0%) had received chelation therapy. Transfusion details for the 12 months prior to enrollment were available for 23 adults, of whom 5 (21.7%) had been regularly transfused (≥6 transfusions) and 18 (78.2%) had not been regularly transfused (0-5 transfusions); in the pediatric cohort (n=24), 8 (33.3%) pts had been regularly transfused and 16 (66.7%) had not. In the prior 12 months, regularly transfused adult and pediatric pts each received an average of about 10 transfusions whereas non-regularly transfused adult and pediatric pts received about 1 transfusion (Table). Additionally, 24/28 (85.7%) adult and 15/29 (51.7%) pediatric pts with FeO had a splenectomy, with median age at splenectomy of 6.5 yrs (range: 1-23 yrs) for the adult cohort and 6.0 yrs (4-12 yrs) for the pediatric cohort. Chelation therapy had been utilized by 20/23 (87.0%) splenectomized adults and 2/4 (50.0%) non-splenectomized adults, while 15/15 (100.0%) splenectomized pediatric pts and 13/14 (92.9%) non-splenectomized pediatric pts had received chelation therapy. Conclusion: These data indicate that FeO is a common complication affecting around 40% of PK deficient pts in this cohort, regardless of age, genotype, transfusion status, or severity. While a large proportion of these pts with PK deficiency and FeO have a high transfusion burden and/or have been splenectomized, FeO still occurs in both adult and pediatric pts without splenectomies, in pts who are not regularly transfused, and even in those who have never been transfused. This analysis supports the need for an initial evaluation and regular monitoring of FeO in all pts with PK deficiency, independent of transfusion status, splenectomy history, or genotype, starting early in life and continuing throughout adulthood. The longitudinal (up to 9 yrs) design of the Peak Registry will allow for continued monitoring and follow-up of FeO and related long-term complications in pts with PK deficiency. Figure 1 Figure 1. Disclosures Bianchi: Agios pharmaceutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Grace: Novartis: Research Funding; Principia: Membership on an entity's Board of Directors or advisory committees; Dova: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios: Research Funding. Glader: Agios: Consultancy. Glenthøj: Novo Nordisk: Honoraria; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Alexion: Research Funding. Kuo: Bluebird Bio: Consultancy; Pfizer: Consultancy, Research Funding; Alexion: Consultancy, Honoraria; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Novartis: Consultancy, Honoraria; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Apellis: Consultancy. Lander: Agios: Other: PK Deficiency Patient Advocacy Advisory Council - patient representative. Layton: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cerus: Membership on an entity's Board of Directors or advisory committees. Viprakasit: Agios: Consultancy, Research Funding; Vifor Pharma: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau; Protagonist Therapeutics: Consultancy, Research Funding; Ionis Pharmaceuticals,: Consultancy, Research Funding; La Jolla Pharmaceuticals: Consultancy, Research Funding. Williams: Agios: Current Employment, Current holder of stock options in a privately-held company. Yan: Agios: Consultancy. McGee: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Van Beers: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Pfizer: Research Funding; RR Mechatronics: Research Funding.

Background: Pyruvate kinase (PK) deficiency is a rare, inherited hemolytic anemia caused by autosomal recessive mutations in the PKLR gene, whereby a glycolytic defect causes a reduction in adenosine triphosphate generation. Current treatment options are supportive and include splenectomy, blood transfusions, and iron chelation therapy. To better understand the natural history, treatment patterns, and burden of disease, the observational PK Deficiency Natural History Study (NHS) (NCT02053480) enrolled 254 adult and pediatric patients with PK deficiency at 30 sites in 6 countries between 2014 and 2017 and followed patients for 2 years. The Peak Registry (NCT03481738) was developed to continue and expand on this research. This retrospective and prospective observational registry aims to enroll 500 adult and pediatric patients at ~ 60 sites in up to 20 countries over 7 years, with 2-9 years of follow-up. Objective: This analysis aimed to characterize the baseline demographics and clinical characteristics of patients with PK deficiency enrolled in the Peak Registry as of 24March2020. Methods: Demographic, diagnostic, medical history, laboratory, treatment, and other relevant data were collected from participating clinicians via electronic case report forms. To be eligible for inclusion in this analysis, patients were required to have genetically confirmed PK deficiency and available demographic information. All analyses reported here are descriptive and based on data as of the date of enrollment in the registry. Continuous variables are summarized by the number of non-missing observations, mean, standard deviation (SD), median, and range. Categorical variables are summarized as counts and percentages. Results: A total of 141 patients met the inclusion criteria, across 11 countries in North America and Europe. A summary of baseline demographics and clinical characteristics is shown in the Table. Fifty patients (35.5%) had completed 2 years of follow-up in the NHS and then moved to the Peak Registry; the remainder were newly recruited to the Peak Registry. The mean age of study participants at enrollment was 25.5 years (SD 19.1); 78 patients (55.3%) were female. Mean reported age at first symptoms was 5.8 years (SD 13.2) and mean age at diagnosis was 11.7 years (SD 16.0). Fifty-seven percent of patients were classified as having missense/missense mutations, 34.4% as having missense/non-missense mutations, and 8.6% as having non-missense/non-missense mutations. The mean hemoglobin at enrollment was 8.8 g/dL (range: 5.8-12.9 g/dL). Mean reticulocyte count was 19.8% (range: 2.2-42.4%), mean lactate dehydrogenase was 382 IU/L (range: 135-849 IU/L), and mean indirect bilirubin was 4.3 mg/dL (range: 0.8-23.1 mg/dL). Among the 45.2% of patients who had been splenectomized, the mean age at splenectomy was 7.2 years. Chelation therapy had been previously prescribed to 40.3% of patients. Among the 27 patients for whom ferritin data were available, the mean was 867.9 ng/L (range: 78.1-2499.0 ng/L), and 18 patients (66.7%) had a level > 500 ng/L. Ninety-nine patients (70.2%) had received at least one transfusion in their lifetime. Among the 45 patients who were known to have received at ≥ 1 transfusion in the 12 months prior to enrollment, the mean number of transfusions during that period was 5 (SD 4.3), with 18 of those patients (40.0%) having received ≥ 6 transfusions. Conclusions: New data emerging from the Peak Registry will provide valuable insights into the patient characteristics, treatment patterns, and burden associated with PK deficiency. The population is demographically heterogenous and represents a broad geography. Patients have a wide range of hemoglobin levels, and iron overload is common. The substantial rates of splenectomy, cholecystectomy, transfusions, and chelation use are indicative of a high disease and treatment burden in patients with PK deficiency. This abstract is presented on behalf of the Peak Registry Steering Committee and Peak Registry Investigators. Disclosures Grace: Novartis: Research Funding; Pfizer: Research Funding; Agios: Research Funding; Dova: Membership on an entity's Board of Directors or advisory committees. Boscoe:Agios Pharmaceuticals: Current Employment, Current equity holder in private company. Bowden:Agios Pharmaceuticals: Current Employment, Current equity holder in private company. Glader:Agios Pharmaceuticals, Inc.: Consultancy. Layton:Cerus: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees. van Beers:Novartis: Research Funding; Pfizer: Research Funding; RR mechatronics: Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees, Research Funding. Yan:Agios Pharmaceuticals: Consultancy. Bianchi:Agios Pharmaceuticals: Other: Scientific Advisor.

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Hexokinase (HK) is a key enzyme of glycolysis, the only metabolic pathway able to provide the red blood cell with ATP. HK deficiency is a very rare hereditary disorder with severe chronic nonspherocytic hemolytic anemia (HNSHA) as a major clinical feature. To date, only 24 patients with HK deficiency have been identified. Here, we report the molecular analysis of six new cases of HK deficiency. A total of six different mutations were detected in HK1, four of them described here for the first time: c.2599C>T p.(His867Tyr), c.1799C>T p.(Thr600Met), c.873-2A>G and c.493-1G>A. The pathogenic nature of the identified missense mutations was confirmed by biochemical and 3-dimensional structural analysis. The effects of the novel splice site mutation c.873-2A>G were studied at the level of pre-mRNA processing, and confirmed at the protein level. All together, these results provide a better insight into the pathogenesis of this rare red cell disorder, and contribute to a better understanding of the genotype-phenotype correlation in HK deficiency.

Background: Transfusion-dependent β-thalassemia (TDT) is associated with several complications necessitating a multidisciplinary approach for diagnosis, treatment and follow-up. Hypogonadism in female TDT patients is one of the most common endocrine complications, requiring hormone replacement therapy (HRT) throughout reproductive life. Little is known about the balance of benefits versus risks of treatment with sex steroids. Aim: The aim of this manuscript is to review the action and the associated adverse effects of HRT in hypogonadal TDT females. Design: Retrospective medical database records from a single centre, over a period of 38 years (January 1980 to June 2018), were reviewed. Study population: Forty-two cases of hypogonadism in TDT females followed in a pediatric and adolescent outpatient clinics, were in included in the study. Methods: Auxological, clinical, laboratory, hormonal and imaging investigations were reviewed, as well as all adverse events registered during HRT. Main results: In general, HRT was safe for most patients. There were few minor side effects and a couple of rare but serious adverse events. Conclusions: The study provides a representative clinical profile of long-term effects of HRT in hypogonadal adolescents and young adult TDT women. Our results highlight also the need for further research in other areas for which HRT may have a role. We hope this will contribute to a wider understanding, and potential improvement, of patient safety and quality of life. (www.actabiomedica.it)

Electrophysiology; Haemolytic anemia; Hereditary spherocytosis Electrofisiologia; Anemia hemolítica; Esferocitosi hereditaria Electrofisiologia; Anèmia hemolítica; Esferocitosi hereditària Congenital haemolytic anaemias are inherited disorders caused by red blood cell membrane and cytoskeletal protein defects, deviant hemoglobin synthesis and metabolic enzyme deficiencies. In many cases, although the causing mutation might be known, the pathophysiology and the connection between the particular mutation and the symptoms of the disease are not completely understood. Thus effective treatment is lagging behind. As in many cases abnormal red blood cell cation content and cation leaks go along with the disease, by direct electrophysiological measurements of the general conductance of red blood cells, we aimed to assess if changes in the membrane conductance could be a possible cause. We recorded whole-cell currents from 29 patients with different types of congenital haemolytic anaemias: 14 with hereditary spherocytosis due to mutations in α-spectrin, β-spectrin, ankyrin and band 3 protein; 6 patients with hereditary xerocytosis due to mutations in Piezo1; 6 patients with enzymatic disorders (3 patients with glucose-6-phosphate dehydrogenase deficiency, 1 patient with pyruvate kinase deficiency, 1 patient with glutamate-cysteine ligase deficiency and 1 patient with glutathione reductase deficiency), 1 patient with β-thalassemia and 2 patients, carriers of several mutations and a complex genotype. While the patients with β-thalassemia and metabolic enzyme deficiencies showed no changes in their membrane conductance, the patients with hereditary spherocytosis and hereditary xerocytosis showed largely variable results depending on the underlying mutation.

Rare anemias and inherited bone marrow failure syndromes are characterized, respectively, by decreased concentrations of hemoglobin or defects in the production of hematopoietic cells leading to single to multiple lineage cytopenias. They are rare and difficult to diagnose due to clinical, cytological and genetic heterogeneity. In this paper, we first address the diagnosis of rare anemias and their causes including marrow failures, erythrocyte defects and disorders of red cell metabolism factors involved in erythrocyte maturation. Finally, we introduce inherited bone marrow failure syndromes and their associated pathologies such as congenital malformations and tumor predisposition, with particular emphasis on the most common diseases: Fanconi anemia, dyskeratosis congenita, Diamond-Blackfan anemia and Shwachman-Diamond syndrome.

Rare anemias and inherited bone marrow failure syndromes are characterized, respectively, by decreased concentrations of hemoglobin or defects in the production of hematopoietic cells leading to single to multiple lineage cytopenias. They are rare and difficult to diagnose due to clinical, cytological and genetic heterogeneity. In this paper, we first address the diagnosis of rare anemias and their causes including marrow failures, erythrocyte defects and disorders of red cell metabolism factors involved in erythrocyte maturation. Finally, we introduce inherited bone marrow failure syndromes and their associated pathologies such as congenital malformations and tumor predisposition, with particular emphasis on the most common diseases: Fanconi anemia, dyskeratosis congenita, Diamond-Blackfan anemia and Shwachman-Diamond syndrome., Las anemias raras y los fallos medulares hereditarios son enfermedades hematológicas caracterizadas, respectivamente, por una disminución de la concentración de hemoglobina o por diversos grados de defectos en la producción de células hematopoyéticas que conducen desde una citopenia de un solo linaje hasta una de múltiples linajes. Son enfermedades raras y difíciles de diagnosticar debido a la heterogeneidad clínica, citológica y genética. En este artículo abordaremos en primer lugar el diagnóstico de las anemias raras y sus causas principales: fallos medulares, defectos del hematíe y trastornos del metabolismo de los factores de maduración eritrocitario. Seguidamente introduciremos los fallos medulares hereditarios y su patología asociada, como son las malformaciones congénitas y la predisposición tumoral, haciendo especial hincapié en los más frecuentes: la anemia de Fanconi, la disqueratosis congénitca, la anemia de Diamond-Blackfan y el síndrome de Shwachman-Diamond.

Pyruvate kinase (PK) deficiency is an autosomal recessive disease caused by mutations in the PKLR gene that lead to reduced red blood cell PK (PK-R) enzyme activity. This rare hereditary glycolytic enzymopathy, with over 300 causative PKLR mutations identified to date, results in defective red blood cell glycolysis and hemolytic anemia. While the clinical presentation is variable, patients with PK deficiency may experience symptoms of hemolytic anemia, most commonly fatigue (sometimes extreme), jaundice, and dyspnea. No disease-specific therapy currently exists and treatment is limited to supportive care, including red blood cell transfusions, splenectomy/cholecystectomy, and iron chelation. Affected neonates may need phototherapy or exchange transfusions for severe hyperbilirubinemia. Allogeneic stem cell transplantation may cure the disease but experience is limited and the outcome is variable. Due the rarity of PK deficiency, its prevalence, clinical burden, and long-term clinical course are not well defined. To address this gap, Boston Children's Hospital is nearing completion of the observational PK deficiency Natural History Study (ClinicalTrials.gov NCT02053480; N=254) to better understand the natural history and clinical burden of the disease. This longitudinal analysis (2-year follow-up) will report on PK deficiency-related signs, symptoms, and treatment outcomes. In order to continue and expand upon the collection of longitudinal data for PK deficiency, the Pyruvate Kinase Deficiency Global Longitudinal Registry (the PEAK Registry; NCT03481738) was developed. This registry study is a global, longitudinal, observational study for adult and pediatric patients with PK deficiency. Its primary objective is to record the natural history, treatment, outcomes, variability in clinical manifestations, and disease burden of patients with PK deficiency. Secondary objectives include data collection to assess the prevalence, incidence, and complications associated with PK deficiency; evaluate pregnancy outcomes; and investigate possible phenotype-genotype correlations. The study also aims to provide longitudinal data to assist physicians with the clinical management of individual patients. In order to maximize the amount of longitudinal data available, a novel data management system is being employed to harmonize Natural History Study and PEAK Registry data. Approximately 500 patients will be enrolled over 7 years at an estimated 60 study centers in up to 20 countries in the 9-year study. All enrolled patients will be followed prospectively for at least 2 years and up to 9 years. Site and patient recruitment began in 2018. As of July 2019, 43 sites in 11 countries are active (Figure) and site recruitment has begun in Thailand, South Korea, and Australia. An update on patient enrollment will be provided. Disclosures Grace: Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding. Bianchi:Agios Pharmaceuticals, Inc.: Consultancy. Glader:Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Glenthøj:Celgene: Consultancy; Novo Nordisk: Honoraria; Alexion: Research Funding; Novartis: Consultancy; Agios Pharmaceuticals, Inc.: Consultancy. Jones:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Kanno:Agios Pharmaceuticals, Inc.: Honoraria. Kuo:Pfizer: Consultancy; Novartis: Consultancy, Honoraria; Celgene: Consultancy; Bluebird Bio: Consultancy; Bioverativ: Other: Data Safety Monitoring Board; Agios: Consultancy; Alexion: Consultancy, Honoraria; Apellis: Consultancy. Layton:Novartis: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Cerus Corporation: Membership on an entity's Board of Directors or advisory committees. van Beers:Pfizer: Research Funding; RR Mechatronics: Research Funding; Agios Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding. Xu:Agios: Employment, Equity Ownership.

Changes in thyroid function and thyroid function tests occur in patients with β-thalassemia major (TM). The frequency of hypothyroidism in TM patients ranges from 4% to 29 % in different reports. The wide variation has been attributed to several factors such as patients' genotype, age, ethnic variations, treatment protocols of transfusions and chelation, and varying compliance to treatment. Hypothyroidism is the result of primary gland failure or insufficient thy­roid gland stimulation by the hypothalamus or pituitary gland. The main laboratory parameters of thyroid function are the assessments of serum thyroid-stimulating hor­mone (TSH) and serum free thyroxine (FT4). It is of primary importance to interpret these measure­ments within the context of the laboratory-specific normative range for each test. An ele­vated serum TSH level with a normal range of serum FT4 level is consistent with subclinical hypothyroidism. A low serum FT4 level with a low, or inappropriately normal, serum TSH level is consistent with secondary hypothyroidism. Doctors caring for TM patients most commonly encounter subjects with subclinical primary hypothy­roidism in the second decade of life. Several aspects remain to be elucidated as the frequency of thyroid cancer and the possible existence of a relationship between thyroid dysfunction on one hand, cardiovascular diseases, components of metabolic syndrome (insulin resistance) and hypercoagulable state on the other hand. Further studies are needed to explain these emerging issues. Following a brief description of thyroid hormone regulation, production and actions, this article is conceptually divided into two parts; the first reports the spectrum of thyroid disease occurring in patients with TM, and the second part focuses on the emerging issues and the open problems in TM patients with thyroid disorders.

Recently, we reported a new mutation of phosphoglycerate kinase (PGK), called PGK-Barcelona, which causes chronic hemolytic anemia associated with progressive neurological impairment. We found a 140T→A substitution that produces an Ile46Asn change located at the N-domain of the enzyme and we suggested that the decrease of the PGK activity is probably related to a loss of enzyme stability. In this paper, by analyzing whole hemolysates and cloned enzymes, we show that both enzymes possess similar kinetic properties (although some differences are observed in the Km values) and the same electrophoretic mobility. However, PGK-Barcelona has higher thermal instability. Therefore, we confirm that the decrease of the red blood cell (RBC) PGK activity caused by the PGK-Barcelona mutation is more closely related to a loss of enzyme stability than to a decrease of enzyme catalytic function. Furthermore, we have measured the levels of glycolytic metabolites and adenine nucleotides in the RBC from controls and from the patient. The increase of 2,3-bisphosphoglycerate and the decrease of ATP RBC levels are the only detected metabolic changes that could cause hemolytic anemia.

Fundamento y objetivo: Las hemoglobinopatias inestables presentan sustituciones de aminoacidos en lugares criticos de la molecula que disminuyen su solubilidad y facilitan su desnaturalizacion y precipitacion. Se describe aqui el primer caso de hemoglobina (Hb) Newcastle en Espana. Caso clinico: Nina de 5 anos que consulto por fiebre de 4 dias. En la exploracion fisica destacaban palidez cutanea y mucosa, subictericia y esplenomegalia. En la analitica sanguinea destacaban: Hb de 79 g/l, hematocrito del 27%, volumen corpuscular medio de 93,4 fl y reticulocitos del 3%. En el frotis de sangre periferica destacaban anisocitosis y policromasia. Resultados: La prueba de estabilidad termica fue positiva. La electroforesis mostro una banda difusa entre HbA2 y HbA. La cromatografia liquida de alta resolucion (HPLC) mostro una Hb no identificada, que correspondia al 12% del total. El estudio molecular demostro la mutacion CD92 His * Pro, Hb Newcastle en estado heterocigoto, patron que se repitio en la madre. Conclusiones: La Hb Newcastle se ha descrito anteriormente en 3 pacientes de origen ingles, ruso y chino. Su expresividad clinica es de anemia hemolitica cronica con crisis de agudizacion tras la ingesta de farmacos oxidantes o infecciones. A diferencia de la electroforesis, la HPLC permite el diagnostico diferencial entre la Hb Newcastle y otras Hb inestables como la Hb Koln.

This paper, prepared by the EFLM Task and Finish Group on Allocation of laboratory tests to different models for performance specifications (TFG-DM), is dealing with criteria for allocating measurands to the different models for analytical performance specifications (APS) recognized in the 1st EFLM Strategic Conference Consensus Statement. Model 1, based on the effect of APS on clinical outcome, is the model of choice for measurands that have a central role in the decision-making of a specific disease or clinical situation and where cut-off/decision limits are established for either diagnosing, screening or monitoring. Total cholesterol, glucose, HbA1c, serum albumin and cardiac troponins represent practical examples. Model 2 is based on components of biological variation and should be applied to measurands that do not have a central role in a specific disease or clinical situation, but where the concentration of the measurand is in a steady state. This is best achieved for measurands under strict homeostatic control in order to preserve their concentrations in the body fluid of interest, but it can also be applied to other measurands that are in a steady state in biological fluids. In this case, it is expected that the “noise” produced by the measurement procedure will not significantly alter the signal provided by the concentration of the measurand. This model especially applies to electrolytes and minerals in blood plasma (sodium, potassium, chloride, bicarbonate, calcium, magnesium, inorganic phosphate) and to creatinine, cystatin C, uric acid and total protein in plasma. Model 3, based on state-of-the-art of the measurement, should be used for all the measurands that cannot be included in models 1 or 2.

The Community added value of Centres of Expertise (CoE) and European Reference Networks (ERN) is particularly high for rare diseases (RD) due to the rarity of these conditions, which implies both a small number of patients and scarcity of expertise within a single country. Gathering expertise at the European level is therefore, paramount in order to ensure equal access to accurate information, appropriate and timely diagnosis and high quality clinical care and follow up for patients with rare diseases. This applies particularly to rare anaemias due to the high number of different rare diseases that constitute this group. In this context, the European Network for Rare and Congenital Anaemias (ENERCA), co-financed by the European Commission, was created in 2002 with the aim of prevention and management of rare anaemias (RA) and the development and promotion of policies to improve the well-being of European Union citizens. The ENERCA White Book is a position paper, developed as a deliverable of the ENERCA (phase 3) project that intends to contribute to the creation of a ERN in RA (ERN-RA) by preparation of the recommendations and, in particular, the definition of the criteria that CoE, local centres (LC) and their interrelations have to fulfil as healthcare providers. It has been nourished by all the activities that have been performed over the past ten years within the ENERCA framework. The White Book is addressed to authorities in charge of the identifying CoE, as an essential requirement for the official recognition of the ERN, to European and national health authorities, Healthcare centres and health professionals, as well as to all other stakeholders interested in RA. It is also addressed to the patients, as a way to empower their community in this process. One particular characteristic of the White Book is the integration of the three main aspects of a CoE: (a) ethical and legal frameworks to ensure the non-discrimination and non-stigmatisation of rare disease patients across Europe, within their sphere of competencies; (b) clinical and laboratory frameworks for defining technical and quality criteria including scope, general and disease specific elements currently defined as technical and professional standards for the diagnosis, treatment and follow-up of patients with RA; and (c) the expectations patients have of CoE. Conceived as a working tool directed to a broad range of stakeholders, the White book has been designed and structured to be comprehensible even to non-technical and /or non-professional audiences. The reader will find an up-to-date description and epidemiological information on RA as well as the European Union background policies for defining CoE and ERN-RA. A working group was created with experts of different profiles, known as the European Working Group on Rare Anaemias (EGRA). In order to achieve its objectives, the methodology used by EGRA, was characterised by three main principles: Interdisciplinary, European coverage, and evidence-based principles. Work has been developed into four sequential steps: 1. Analysis of the current situation of RA in Europe by healthcare professionals in order to identify the most relevant issues that have to be addressed by a centre in order for it to be recommended as CoE. 2. Preparation of questionnaires to perform surveys on how the relevant issues identified in step 1 can be translated into practical recommendations. 3. Analysis of the questionnaire results by face to face meetings, feedback and consensus evaluation, and 4. Preparation of a report on ENERCA policy recommendations for CoE. This report is presented in a user-friendly format, easy to understand and available through the ENERCA website (www.enerca.org). Several important conclusions can be drawn from the ENERCA White Book, including the importance of laboratories involved in the diagnosis of RA, patient oriented and multidisciplinary care at the CoE, the need for coordination and cooperation within and outside the centre, the provision of information to patients and health professionals and the involvement of public authorities at the national and European levels. Official recognition of this structure and assurance of its long term sustainability will only be achieved if public authorities work hand by hand with both professionals experts in different disciplines and patients. Finally, the ENERCA White book aims to be a practical tool for health authorities of Member States (MS) that are preparing their national directory of formally designated CoE. For this, it is important that MS authorities recognise RA as an important health component to be included within the National Plans or Actions for Rare Diseases.

Background: Major haemoglobinopathies (MH), such as thalassaemia syndromes (Thal) and sickle cell disorders (SCD), are genetic defects associated with chronic anaemia and other complications. In Europe, MH are rare diseases (RD) but their prevalence is significantly growing in many countries due to mobility and migration flows. This creates a growing health problem in the EU that has not yet been effectively addressed by Member States (MS) authorities. The present study has been conducted with the aim of: (i) providing an overview of policies for MH in 10 EU member states (MS) (ii) analysing the challenges linked to these RD due to growing requirements imposed by population, mobility and migration trends and (iii) identifying gaps, proposing improvements on existing policies, or developing new ones to fit the identified needs. Methods. The study has been undertaken by a group of members of the European Network for Rare and Congenital Anaemias (ENERCA) and the Thalassaemia International Federation (TIF), in collaboration with the public affairs firm Burson-Marsteller Brussels. Data from 10 EU countries have been gathered using targeted desk research and one-to-one interviews with local stakeholders, including healthcare professionals, patients and public health officers/providers. Results: 1. MH are the most common RD in all the 10 countries, 2. Data on prevalence, overall burden, trends, and clinical follow up costs are lacking in most countries. 3. Neonatal screening practices show a wide variation across and within countries. 4. Awareness on MH and their related complications is very low, exception made of Italy, Greece, Cyprus and UK, 5. No disaggregated data is available to understand the impact of mobility and migration on the prevalence of haemoglobinopathies, and how healthcare delivery systems should adapt to respond to this situation. 6. Targeted policy measures and/or actions are generally lacking and/or delayed. Conclusions: Ten policy recommendations have been drawn from this study, building on 2006 WHO recommendations for MH to include haemoglobinopathies in National Plans of Actions for Rare Diseases. © 2014 Aguilar Martinez et al. licensee BioMed Central Ltd., SCOPUS: re.j, info:eu-repo/semantics/published

In 1972, Valentine et al described, under the name of ‘non-spherocytic haemolytic anaemia, high red cell ATP and ribose phosphate pyrophosphokinase (RPK; EC 2.7.6.1) deficiency’, an obscure congenital haemolytic anaemia with the characteristic feature of red blood cell basophilic stippling. The activity of Embden–Meyerhof pathway and hexose monophosphate shunt were normal, and the concentrations of reduced glutathione and of ATP were raised 2 SD above the normal mean. The low values of RPK also encountered were considered to be an epiphenomenon rather than a causative defect. One year later, further studies performed in two new kindreds with the same haemolytic disorder associated with persistent basophilic stippling were described under the name of ‘haemolytic disorders associated with increased ATP’. In 1974, two new and important observations contributed to the final identification of the disease: the patients' red blood cells (RBCs) contained large amounts of nucleotides (pyrimidine nucleotides), and in all cases they were deficient in a hitherto unrecognized enzyme called pyrimidine 5′ nucleotidase (P5N). In conclusion, all these cases were formerly referred to as ‘high ATP syndromes’ because of the erroneous assumption that the large number of nucleotides within deficient RBCs were adenine phosphate rather than pyrimidine phosphate. Twenty-five years after its description, P5N deficiency has been reported in about 35 unrelated families from different parts of the world, and it has become one commonly identified cause of hereditary non-spherocytic haemolytic anaemia due to RBC enzymopathy. Genetic transmission is via the autosomic recessive mode, and only homozygous or compound heterozygous are clinically affected. Family members who are biochemically heterozygous are haematologically normal and difficult to detect. Unfortunately, the precise gene mutation or mutations causing the disease remain unknown.

With the development of sophisticated automated blood cell analyzers, the proportion of blood samples that require a blood smear examination has steadily decreased. Microscopic examination of stained blood smear, however, still remains a crucial diagnostic aid if an automated instrument produces a highly improbable or doubtful result or abnormal cell flags and it is, in general, performed from anticoagulant whole blood according to standardized procedure.Because of transfer process of collected blood samples the it is of utmost importance to analyse and recognize the effect of EDTA-anticoagulated whole blood storage on cell morphology examination.

Migration from different parts of the world to several European countries leads to the introduction of haemoglobinopathy genes into the population, which creates several demanding needs for prevention and treatment services for Hb disorders. In this paper we examined the degree to which European health services have responded to such challenges and in particular to health services necessary to address the needs of patients with thalassaemia and sickle cell disease (SCD). Information on available services was obtained from international organizations, collaborated European project, and the Thalassaemia International Federation (TIF) Databases, which include information from published surveys, registries, field trips, and delegation visits to countries and regions by expert advisors, local associations, and other collaborators’ reports. Results show that countries with traditional strong prevention and treatment programs are well prepared to face the above challenges, while others are urgently needed to address these problems in a systematic way. The Thalassaemia International Federation (TIF) is committed to monitor the progress, raise awareness, and support the promotion of more immigrant-oriented health policies to ensure their integration in society and their access to appropriate, adequate, and timely health services.

Previous studies found that ADA is present on the surface of mononuclear blood cells from healthy patients. Because the expression of this surface antigen depends upon the cell type, the presence of ADA on the plasma membrane of cells from patients with malignant hematologic diseases was studied by flow cytometry. The highest percentage of expression was found in CLL, whereas the lowest was found in T-cell-derived malignancies. The enzyme expression in immortalized cell lines showed a similar pattern, with the highest expression (95% +/- 5%) in the SKW64 B-derived cell line, the lowest (15% +/- 5%) in Jurkat T-lymphoma derived cells, and the intermediate (32% +/- 8%) in K562 cells derived from a chronic myelogenous leukemia. Double labeling ADA/CD5 and ADA/CD19, as well as the correlation of ADA expression with the expression of other surface markers, indicate that surface ADA might be considered a novel marker for CLL.

Registry and epidemiological data of Rare Anaemias (RA) in Europe is in general still incomplete and/or partially documented. One important issue is the increasing prevalence of haemoglobin disorders (HD) due to migrations from high prevalence areas. The size of the problem, particularly for sickle cell disease (SCD), is already having an impact on health services in many European countries. The best known cause of rare anaemias associated with congenital haemolytic anaemia (CHA) in Europe is Hereditary Spherocytosis (HS) a red blood cell (RBC) membrane defect with a prevalence of 1 to 5 cases per 10.000 individuals. Some other causes of CHA are extremely rare and only few individual cases have been described worldwide (i.e. some RBC enzymopathies). Congenital defects of erythropoiesis are less frequent Diamond-Blackfan Anaemia (DBA) and Fanconi Anaemia (FA) exhibit a very low prevalence ranging from 4 to 7 per million live births. Congenital Dyserythropoietic Anaemia (CDA), a genetically heterogenous group, is still less frequent and exhibits a large variability of frequency depending on the European region: 0.1-3.0 cases per million births In addition many cases are known from a large autosomal dominant family in Sweden. Although incidence of Paroxysmal Nocturnal Haemoglobinuria (PNH) in Europe is still unknown, data collection from different sources has given quotes of 1 case per 100,000 individuals to 5 cases per million births.

Registry and epidemiological data of Rare Anaemias (RA) in Europe is in general still incomplete and/or partially documented. One important issue is the increasing prevalence of haemoglobin disorders (HD) due to migrations from high prevalence areas. The size of the problem, particularly for sickle cell disease (SCD), is already having an impact on health services in many European countries. The best known cause of rare anaemias associated with congenital haemolytic anaemia (CHA) in Europe is Hereditary Spherocytosis (HS) a red blood cell (RBC) membrane defect with a prevalence of 1 to 5 cases per 10.000 individuals. Some other causes of CHA are extremely rare and only few individual cases have been described worldwide (i.e. some RBC enzymopathies). Congenital defects of erythropoiesis are less frequent Diamond-Blackfan Anaemia (DBA) and Fanconi Anaemia (FA) exhibit a very low prevalence ranging from 4 to 7 per million live births. Congenital Dyserythropoietic Anaemia (CDA), a genetically heterogenous group, is still less frequent and exhibits a large variability of frequency depending on the European region: 0.1-3.0 cases per million births In addition many cases are known from a large autosomal dominant family in Sweden. Although incidence of Paroxysmal Nocturnal Haemoglobinuria (PNH) in Europe is still unknown, data collection from different sources has given quotes of 1 case per 100,000 individuals to 5 cases per million births.

The immunophenotype of leukaemia cells from 60 patients with acute myeloid leukaemia (AML) was analysed with the APAAP technique using a panel of anti-myeloid and lymphoid associated monoclonal antibodies (McAb). Cells from all cases, including three with negative cytochemical features, were labelled by at least one of the anti-myeloid McAb CD13, anti-myeloperoxidase (anti-Mpo), and/or CD14. The most sensitive marker was CD13, since it was positive in 90% of cases. In two out of three AML cases defined as M0-AML, CD13 was expressed in the cytoplasm but not on the membrane; in these three cases peroxidase (Mpo) was not detected by conventional cytochemistry, but could be demonstrated in all of them using the McAb anti-Mpo. The simultaneous expression of CD14 and CD68 McAb was often confined to the M4 and M5 FAB AML subtypes (92% cases) as compared to the others: M1, M2, M3 (18% cases). Lymphoid antigens were rarely positive (TdT+: 13%, CD7+: 15%, CD19+: 5%) and none of the AML cases were CD3+ or CD10+. By contrast, CD4 was expressed in blasts from 44% of cases and this was not restricted to AML with a monocytic component (M4, M5) but also found in other subtypes. There were no significant differences in the clinical or prognostic features according to the positivity or negativity with TdT and CD4. By contrast, expression of CD7 was associated with refractoriness to the treatment or short complete remission duration, although the number of patients is too small to draw firm conclusions. Our findings support the clinical and diagnostic relevance of immunophenotypic studies in AML.

In the last 20 years, migratory flows have changed the pattern of beta-thalassemia (beta-thal) mutations in Catalonia and have also increased beta(S) prevalence, either alone or in association with beta-thal alleles. Characterization of the beta gene is needed for genetic counseling for beta-thal major and also for sickle cell diseases. The purpose of this study was to investigate the current distribution pattern of beta-thal mutations. Seventy nine individuals were characterized at the molecular level. As a first step, frequent mutations in the Mediterranean region were screened and when none of these mutations were identified, the beta-globin gene was sequenced. Screening for common mutations allowed the characterization of 60 individuals. In the remaining 19 cases, 11 different mutations were identified. beta-Thalassemia heterogeneity in Spain has markedly increased, leading to the requirement of including new methods for genetic diagnosis. Prevention of beta-thal major and sickle cell disease are necessary since their prevalence in Spain is increasing dramatically.