Abstract Objectives SCD negatively impacts patient’s health-related quality of life (HRQoL). The ASCEND study investigated how SCD impacts the HRQoL of adults in Portugal, focusing on its physical, emotional, and social burdens. Methods This non-interventional, cross-sectional study included two cohorts of adult SCD patients from seven Portuguese Reference Centers (Cohort 1) and the Portuguese Patient Association (Cohort 2). Sociodemographic and patient-reported outcomes were collected for both cohorts, along with clinical data for Cohort 1, between February and September/2022. Results 211 adult SCD patients (Cohort 1: 200; Cohort 2: 11) were included (median age of 33.0 years, 58.8% male). Nearly 90% reported complications. Patients were diagnosed at a median age of 2.0 years, mainly with the HbSS variant (89.0%), and 86.0% were under treatment (73.5% on hydroxyurea, 45.0% on chronic transfusion). All had lifetime pain episodes, with 72.6% experiencing at least one in the previous year. Most (91.9%) adopted daily strategies to prevent pain episodes, 67.1% had pain management plans, and 46.7% self-managed crises at home. Pain/discomfort (60.6%) and anxiety/depression (51.0%) were major problems (median EQ-5D-5L score = 0.91), affecting emotional well-being (53.8%) and/or social life (49.0%). Indeed, less frequent pain significantly correlated with improved HRQoL (P = .001). While 40.0% felt neglected, 70.5% rarely or never experienced solitude, and 84.6% reported a sense of support. Conclusion The ASCEND study reveals the multifaceted impact of SCD on adults in Portugal and its influence on HRQoL, emphasizing the need for a multidisciplinary care approach and effective self-management education to improve patient outcomes.

β-thalassemia is a globally distributed hereditary red blood cell disorder. Up to now, clinical management of transfusion-dependent β-thalassemia (TDT) patients is still based on chronic transfusion combined with iron chelation therapy. Allogeneic hematopoietic cell transplantation potentially provides a cure, but few patients have an HLA-identical sibling, and optimal results are reported in patients ≤ 14 years. The European Hematology Association (EHA), through the EHA Scientific Working Group on Red Cells/Iron and the European Bone Marrow Transplantation (EBMT) group, has updated a 2021 EHA decision-making algorithm on evidence and expert consensus with the aim of identifying which patients with TDT could benefit from gene therapy (GT). Indeed, it is important to establish the patient setting for whom it is a priority, particularly in the early phase of real-world use outside experimental trials. Moreover, actual price, limited availability, and resource disposal constitute a further indication of a rational and progressive approach to this innovative treatment. In this expert consensus document, different clinical scenarios have been considered and analyzed for the possible impact on treatment outcome. This expert opinion provides dynamic, updatable, priority-based guidance for physicians taking care of TDT patients.

Sickle cell disease (SCD), one of the most prevalent genetic disorders worldwide, remains a significantly neglected public health issue in Portuguese-speaking countries, despite an estimated 1 million affected individuals. To address it, the Lusophone Specialists’ Alliance in Sickle Cell Disease (ALUA) was established and organized the 1st International Conference on Sickle Cell Disease in Lusophone Countries. Held on June 19, 2024 and hosted by the CPLP in Lisbon, Portugal, the event brought together stakeholders from 7 countries to assess current challenges and define shared priorities for action and collaboration. A structured survey was conducted among 29 participants involved in SCD care across 7 Lusophone countries. The questionnaire addressed diagnostics, treatment, workforce, registries, and policies. Its findings were presented to delegations from the Lusophone countries in attendance, discussing them along with other international recommendations. Findings revealed widespread disparities in newborn screening, treatment access, and specialist availability. Only Brazil and Portugal conduct universal newborn screening for SCD, while point-of-care diagnostics are beginning to improve early detection in other countries. Access to hydroxyurea is inconsistent, and blood transfusion safety varies. Participants from all countries reported a lack of trained professionals and SCD in medical curricula. Structured transition to adult care and national data systems are also missing in many contexts. Conference attendants identified 3 shared priorities: raising awareness of SCD through education and advocacy; strengthening data, screening, and follow-up systems while acting without delay; and ensuring equitable access to essential care and context-adapted clinical guidance across the Lusophone world.

Thalassemia and sickle cell disease (SCD) are among the most common monogenic disorders worldwide. They cause chronic hemolytic anemia, the consequences and prognosis of which vary considerably depending on the genetic characteristics of patients and the healthcare system in their country of residence. Both diseases are autosomal recessive in their transmission, with carriers generally being asymptomatic. Informing carriers of thalassemia or SCD about reproductive risks and choices, while taking into account cultural and religious considerations, is a priority within global strategies to improve outcomes for these diseases. The European Hematology Association (EHA)'s Topic In Focus (TIF) Hemoglobinopathies Group created a focus group of hematologists, patients, anthropologists, and an obstetrician from Europe, the Middle East, India, and Africa. The Group considered that preconceptual screening tests would correspond to tests conducted before pregnancy (screening for carriers before marriage/conception), antenatal screening referred to tests completed on pregnant women, and prenatal diagnosis referred to tests performed on the fetus. It proposed guidelines addressing optimal timing of screening, appropriate laboratory tests, and communication strategies, taking into account the great diversity of regions and cultures where thalassemia and SCD are present. A main discussion point was that no recommendations would be given for couples about reproductive decisions, and that the aim was to present the existing and available options in different countries. Eight questions were examined using available literature, leading to the formulation of seven recommendations, which were submitted to a vote using the Delphi method. Consensus agreement was obtained for all recommendations.

BACKGROUND:While an increase in fetal hemoglobin (HbF) has no consequences in healthy adults, clinical benefits can be promoted in sickle cell disease (SCD) and β-thalassemia patients. Single-nucleotide polymorphisms (SNPs) in three genomic regions: the HBB gene cluster, the BCL11A gene, and the HBS1L-MYB (HMIP) intergenic region, have been associated with HbF regulation. Therefore, the present study aimed to examine the potential association of SNPs in BCL11A (rs11886868 and rs1427407), HMIP (rs66650371 and rs4895441), HBG2 (rs7482144), and BGLT3 (rs7924684) with HbF levels in an adult population sample from São Tomé e Príncipe (Central Africa). METHODS:A total of 145 women aged 18 to 49 years were involved in this study, comprising 98 women with the normal hemoglobin (Hb) genotype (HbAA) and 47 with sickle cell trait (HbAS). From the HbAA individuals, we selected a control group of 60 subjects with normal HbF levels, ranging from 0.2% to 1.4% (mean: 0.75%), and a case group of 38 subjects with elevated HbF levels, ranging from 1.8% to 3.7% (mean: 2.35%). In the group of HbAS individuals, the HbF levels ranged from 0.4% to 3.7% (mean: 1.56%). SNP genotyping was conducted using standard molecular methods. RESULTS:Logistic regression, in the additive model, revealed significant associations with increased levels of HbF for the minor alleles of the two BCL11A SNPs, rs11886868 [C] and rs1427407 [T], in HbAA women (p = 0.00018 and p = 0.00076, respectively). When comparisons of HbF levels were conducted among genotypes in the HbAA women, significant differences were observed for BCL11A SNPs rs11886868 and rs1427407, as well as for the HBG2 rs7482144 and BGLT3 rs7924684 variants. We found no association between HbF levels and the two HMIP variants rs66650371 and rs4895441 in the HbAA women. Among the HbAS women, no statistically significant associations were observed between the six analyzed polymorphisms and HbF levels (p > 0.05). CONCLUSIONS:We successfully replicated the association between the two well-known BCL11A SNPs, rs11886868 and rs1427407, with HbF levels in women with the normal HbAA genotype from São Tomé e Príncipe. Other signals of association with HbF levels were identified for the SNPs HBG2 (rs7482144) and BGLT3 (rs7924684).

BackgroundThe use of social media platforms for sharing health-related information is on the rise. Sickle cell disease (SCD) affects millions of people worldwide. However, discussions by SCD stakeholders on social media remain unexplored. This study aimed to analyze discussions among SCD stakeholders on social media to understand their awareness of SCD and to explore their perceptions of the patient journey, hospitalizations and complications due to SCD, the impact of the disease on quality of life (QoL), and current unmet needs by using social media listening (SML).MethodsData was retrospectively collected from April 2019 to April 2021 on SCD specific terms in 14 European countries from blogs, forums, and social networking sites (Twitter, public Facebook, YouTube, and Instagram). Advanced social media analytics tools, Talkwalker and Social Studio, were used for data aggregation and analysis. Conversations were filtered and contextualized through a 3-tier technique involving automated relevancy algorithms and manual review.ResultsOf 317.9K conversations on SCD (93% Twitter), 945 posts on relevant patient-centric conversation were analyzed. Most patients were females (73%) and ≤30 years old (75%). Patient journey stages were addressed in 52% of conversations. Patient journey conversations were mainly regarding symptoms (56%) (mainly pain episodes, pain in general, and vaso-occlusive crises) and treatment (44%). Conversations on hospital visits or hospitalization mostly revolved around crises faced due to symptoms. Impact on QoL, especially emotional impact (56%), was also extensively discussed. Unmet needs were derived from 24% of the conversations, lack of awareness of SCD (42%) and lack of empathy and support from HCPs (24%) being the most frequent topics. Patients reported having their symptoms questioned or dismissed by healthcare professionals, which they attributed to racial bias.ConclusionSML proves to be a useful tool for exploring the real experiences, concerns, and needs of SCD patients and other stakeholders. Analysis of SCD-related social media posts reveals that discussions mainly focus on symptoms, particularly pain, treatment, and the emotional impact of SCD on QoL. These insights are crucial for enhancing the management of SCD patients.

The Portuguese Newborn Screening Program currently includes 28 pathologies: congenital hypothyroidism, cystic fibrosis, 24 inborn errors of metabolism, sickle cell disease and spinal muscular atrophy. This pilot study for sickle cell disease newborn screening, including 188,217 samples, was performed between May 2021 and December 2023, with phase I, including 24,130 newborns, in the Lisbon and Setubal districts and phase II, including 164,087 newborns, in the whole country. DBS samples were analyzed through capillary electrophoresis. In phase I, a high birth incidence of sickle cell disease was found (1:928 NBs), resulting from the identification of 24 HbSS and 2 HbSC patients. This birth incidence decreased but remained significant when the pilot study for sickle cell disease newborn screening was expanded to a national level, with the identification of 67 sickle cell disease patients (59 HbSS and 8 HbSC), revealing a birth incidence of 1:2449 NBs. These data suggest that this condition is becoming increasingly relevant in Portugal, thus reflecting a general European trend, where sickle cell disease is already recognized as a public health problem. Therefore, it highlights the importance of its integration into the Portuguese National Newborn Screening Program panel in January 2024, thus allowing the early identification and clinical follow-up of these patients.

Kruppel-like factor 1 (KLF1) is an essential erythroid-specific transcription factor. Several reports have shown that KLF1 gene mutations are associated with increased levels of Hb F and Hb A2. However, scarce population studies have analysed common KLF1 variations. This study examines the potential association with Hb F and Hb A2 levels in β-thalassemia (β-thal) carriers of Portuguese ancestry of the four common KLF1 gene variants: −251C>G (rs3817621) and −148G>A (rs79334031), in the promoter region; and c.115A>C (p.Met39Leu) (rs112631212) and c.304T>C (p.Ser102Pro) (rs2072597), in exon 2. Ninety-two Portuguese β-thal carriers (43 males and 49 females) aged 2 to 77 years old (mean 32.55 years) were engaged in the study. Hb F levels range from 0.2 to 12.5

Thalassemia is one of the most prevalent monogenic disorders in low- and middle-income countries (LMICs). There are an estimated 270 million carriers of hemoglobinopathies (abnormal hemoglobins and/or thalassemia) worldwide, necessitating global methods and solutions for effective and optimal therapy. LMICs are disproportionately impacted by thalassemia, and due to disparities in genomics awareness and diagnostic resources, certain LMICs lag behind high-income countries (HICs). This spurred the establishment of the Global Globin Network (GGN) in 2015 at UNESCO, Paris, as a project-wide endeavor within the Human Variome Project (HVP). Primarily aimed at enhancing thalassemia clinical services, research, and genomic diagnostic capabilities with a focus on LMIC needs, GGN aims to foster data collection in a shared database by all affected nations, thus improving data sharing and thalassemia management. In this paper, we propose a minimum requirement for establishing a genomic database in thalassemia based on the HVP database guidelines. We suggest using an existing platform recommended by HVP, the Leiden Open Variation Database (LOVD) (https://www.lovd.nl/). Adoption of our proposed criteria will assist in improving or supplementing the existing databases, allowing for better-quality services for individuals with thalassemia. Database URL: https://www.lovd.nl/.

ABSTRACT: Sickle cell anemia occurs in individuals who are homozygous for the sickle hemoglobin (HbS) allele. The haplotypes of the HbS alleles can be traditionally ascertained through the analysis of five to eight informative restriction fragment length polymorphisms within the HBB gene cluster, defining the five major HbS haplotypes, commonly referred to as Bantu or Central African Republic (CAR), Benin (BEN), Cameroon (CAM), Senegal (SEN), and Arab-Indian. In a recent study, it was suggested that four single nucleotide polymorphisms (SNPs)—rs3834466, rs28440105, rs10128556, and rs968857—are sufficient to characterize most of the HbS haplotypes. In the present study, we investigated the HbS haplotypes in a population sample from São Tomé e Príncipe (Central Africa) by analyzing this set of four SNPs. Additionally, we included the HBG2 rs7482144 (−158C>T) SNP, which is commonly used in the classical HbS haplotype classification, to compare the HbS haplotypes derived from the two different plex systems. Blood samples of 50 heterozygous (HbAS), nine homozygous (HbSS), and three normal HbAA individuals were engaged in this study. Genotyping was performed by the polymerase chain reaction–restriction fragment length polymorphism method. Haplotypes were derived using the Arlequin software. In the 4-plex system, seven different haplotypes were inferred among the 68 HbS chromosomes, corresponding to CAR, 36.8%; BEN, 25.0%; CAM, 13.2%; SEN, 11.8%; and three different sets of unknown HbS haplotypes, 11.4%. The further inclusion of rs7482144 (XmnI) led to the inference of a total of eight distinct HbS haplotypes as follows: CAR, 36.8%; BEN, 25.0%; CAM, 13.2%; SEN, 8.8%; and four different clusters of unknown HbS haplotypes, 16.2%. Using only the nine homozygous HbSS individuals, the same five different haplotypes were identified for both systems, with all common haplotypes occurring at equal frequencies: CAR, 27.8%; BEN, 38.9%; CAM, 16.7%; and SEN, 11.1%. One atypical haplotype was identified at a frequency of 5.6%. Our findings suggest that the 4-plex system may not be entirely accurate in identifying the SEN haplotype when considering heterozygous individuals for the HbS allele in the study population. Additionally, the haplotype background of HbS observed in the S. Tomean population provided further insights compared with previous reports.

Background: Ultra-rare anemia disorders (uRADs) are commonly neglected from health planning and clinical research hampering its timely diagnosis and leading to sub-optimal clinical management and very few treatment options. The Rare Anemia Disorders European Epidemiological Platform (RADeep) together with the ERN-EuroBloodNet have joint efforts to establish the uRADAR initiative aiming to develop a referral frame for patients affected by uRADs in the European Union. The uRADAR ultimate goal is to enable access to clinical trials (CT), including drug repurposing i.e. ERN-EuroBloodNet SATISFY Phase 2 Trial (NCT05935202). Methodology: The uRADAR disease scope includes all ultra-rare hemolytic anemias (pyruvate kinase deficiency (PKD), other rare cell enzyme and membrane defects, congenital dyserytropoietic anemias I-IV (CDAs), unstable hemoglobinopathies, ultra-rare iron metabolism defects, sideroblastic anemias and metheglobinemias. Chronic and severe forms of hereditary spherocytosis (HS) and G6PD deficiency were also considered. The RADeep uRADAR module for standardized collection of uRADs data was developed in a REDcap web application. We gathered disaggregated data by age ranges (0-11, 12-15, 16-17 and ≥18 yo) on: sex, genetic diagnosis, common CT exclusion criteria, and therapeutic intervention (splenectomy and/or blood transfusion dependence (TD)). Anemia is considered as Hb <11g/dL in NTD patients. Severity based on therapeutic intervention and anemia is analyzed for >12yo. Results: Data from 5,623 patients from 82 centers (13 EU countries, Norway and United Kingdom) has been collected, 1,302 (23%) of them with an uRAD. The cohort has a balanced age and sex distribution, except for X-linked diseases. Information about severity is available for 3,465 (62%) patients, 1,015 (78%) for the uRADs. Genetic confirmation is available for 815 (14%) patients, 308 (24%) for the uRADs. Analyzing by age ranges, we noted patients were constantly diagnosed and followed during pediatric care, however about 2/3 are lost during follow-up at adult age. RBC enzyme defects: 1,600 patients (1,175 G6PD deficiency, 415 PKD and 46 Other). Even G6PD deficiency is a non-severe disease in most cases we detected 38.4% anemic patients. In PKD 48% required therapeutic intervention. 26.7% were anemic and 10.3% remained TD after splenectomy, twice as reported for other ultra-rare enzyme defects. Membranopathies: 3,468 patients (3,146 HS, 162 Hereditary elliptocytosis-HE, 122 dehydrated hereditary stomatocytosis-DHS and 38 Other). In the HS group 46% required therapeutic intervention and 26.4% were anemic. Only 3 patients (0.27%) remained TD after splenectomy. HE is usually not severe, nevertheless 9% required therapeutic intervention and 21.6% were anemic. In DHS 11.3% were anemic. CDAs: 154 patients. 45% required therapeutic intervention. 18% were anemic. 6.4% remained TD after splenectomy. Ultra-rare iron defects: 46 patients. 6% required therapeutic intervention. 6.3% were anemic. 2.9% remained TD after splenectomy. Sideroblastic anemias: 84 patients. 24% required therapeutic intervention. 5.3% were anemic. 6% remained TD after splenectomy. Interestingly we also collected 21 patients with sitosterolemia, 175 unstable hemoglobinopathies and 60 with congenital methemoglobinemia. These numbers are higher than expected suggesting an under-representation of these diseases in available literature. Conclusions Only 24% of uRADs patients had confirmed molecular diagnosis thus precluding the inclusion in clinical trials for about 2/3 of patients. Although splenectomy is the main therapeutic option for most uRADs, we detected 47 patients over 12yo who are non-splenectomized despite being TD. About 2/3 of adult patients are lost in follow-up which is concerning due to the majority of serious complications appearing in this period and the cutoff age for inclusion in clinical trials is usually 18yo. Overall only 128 (2%) patients presented with the usual exclusion criteria from clinical trials. There is a need for national health policies that support the concentration of patients affected by uRADs in expert centers for both diagnosis and follow-up. Currently, patients with uRADs are spread across EU, hampering enrollment in clinical trials. The uRADAR approach represents a valid approach to retrieve information on patient's severity and distribution.

Background Sickle Cell Disorder is Africa’s most prevalent genetic disease. Yet, it remains a neglected condition, with high mortality under-five, and a lack of population-based studies in the region. This is the first of its kind in São Tomé e Príncipe, aiming to estimate the prevalence of sickle cell trait and other haemoglobin variants in women of reproductive age and its associated factors. Methods We conducted a cluster survey in 35 neighbourhoods. Haemoglobin was assessed through point-of-care capillary electrophoresis or high-performance liquid chromatography, and sociodemographic data through questionnaires. The weighted prevalence of sickle cell trait (HbAS) and HbC carriers was estimated with a 95% confidence interval (95% CI). We calculated weighted prevalence ratios (95% CI) through robust Poisson regression for its association with age and individual and collective genetic heritage. Findings The prevalence of sickle cell trait in women of reproductive age in São Tomé e Príncipe ( n = 376) was 13.45% (95% CI: 9.05-19.00). The prevalence of HbC carriers was 8.00% (95% CI: 4.71-12.00). Older age and speaking Forro or Angolar were positively associated with having sickle cell trait. Interpretation The prevalence of sickle cell trait in São Tomé e Príncipe ranks high in the West African region. The country should follow international guidelines, implementing newborn screening and comprehensive healthcare management.

*Gazi University Faculty of Medicine, Pediatrics Hematology, Ankara, Turkey †Laboratório de Hematologia Molecular Hospital, Pediatrico, Portugal This article does not contain any studies with human participants or animals performed by any of the authors. Informed consent was obtained from the patient and his parents. The authors declare no conflict of interest. Reprints: Zühre Kaya, MD, Department of Pediatrics, Unit of Pediatric Hematology, Gazi University School of Medicine, Beşevler, Ankara, Turkey 06500 (e-mail: [email protected]).

Submitting Novel Globin Gene Variants to HemoglobinIt is now customary to report genetic variants in genomewide and locus/disease-specific databases.Different databases have different features, which are quite useful to the scientific community.At present, HbVar (http://globin.bx.psu.edu/hbvar [1]; established in 2000, as a continuation of Titus Huisman's Syllabi for Hemoglobin Variants and Thalassemia mutations and IthaGenes (https://www.ithanet.eu/db/ithagenes [2] established in 2007, along with the recently released IthaPhen (https://www.ithanet.eu/db/ithaphen[3], are disease-specific databases for the documentation and annotation of variants in the globin gene clusters and their associated phenotype.HbVar encourages variants to be submitted to the database after being published in the literature [4].The advantage is that diagnostic support for a certain variant's pathogenicity is peer-reviewed and hematological data are available for consultation.This is to prevent some variants from ending up as personal communications without any supportive evidence.Nevertheless, after the publication of the micro-attribution approach in 2011, HbVar also welcomes submissions of novel variants even before they are published in the literature.In this case, the review of the supporting evidence is more stringent.The IthaGenes and ClinVar [5] policy is to encourage early submission of novel variants to enable early access to such knowledge, even if unpublished, as this may sometimes take years to be published or never happen.A working group of experts, recruited from HEMOGLOBIN's editorial board, has discussed this issue, and proposed the following structured way to present novel variants to

Topic: 26. Sickle cell disease Background: Sickle cell disease (SCD) is an autosomal recessive blood disorder characterized by abnormal hemoglobin formation, leading to a more rigid and sickle-shaped red blood cells. Both acute and chronic complications of SCD are of extreme impact on the patients and their families. Hence, the occurrence of painful vaso-occlusive crises (VOC) is a hallmark of the disease. Still, other complications also arise from SCD that significantly impact patient’s health-related quality of life. Aims: Characterization of the demographic and clinical characteristics of SCD patients living in Portugal. Methods: Non-interventional cross-sectional study of adult patients with a confirmed diagnosis of SCD conducted in Portugal, from February to September 2022. Patients were ether recruited from participating hospitals, in a real-world setting (cohort 1), or from a patient association group (cohort 2). Primary data (clinical information) were only available for cohort 1, whereas secondary data (demographic information) were collected from both cohorts. Results: 211 SCD patients participated in this study. Most were female (58.8%), with a mean age of 34.0±11.2 years, ranging from 18 to 67 years, and normal weight (body mass index [BMI]=22.1±3.7 Kg/m2). 69.7% of patients lived in the Lisbon metropolitan area. They were mainly Portuguese (56.4%) and most of their parents originated from Angola (mother: 54.8%; father: 51.0%). Only 9.5% and 8.1% of the patients had a Portuguese-born mother or father, respectively. Patients were diagnosed at a mean age of 5.4±9.1 years and predominantly had the HbSS subtype (89.0%). Regarding acute complications of SCD, in the latter year, patients experienced a mean of at least 2.0±1.6 VOC episodes, of which 72.7% had one or more. The number of VOC increased with decreasing patient’s BMI (ρ=-0.142, p=0.045) and was age-independent. As for comorbidities, at least one was present in 89.5% of patients, hepatobiliary disorders (26.0%) and splenic sequestration (26.0%) being the most frequent. Patients aged ≥45 years (<45 years: median [IQR]=2.0 [1.0;3.0]; ≥45 years: 3.0 [2.0;4.0]; p=0.006) had more chronic comorbidities. The vast majority (85.5%) of patients received treatment and/or specific procedures for SCD: 73.5% were on hydroxyurea (HU) therapy and 44.5% received blood transfusions. Additionally, concomitant medication was prescribed to 97.0% of patients, namely antianemics (90.5%), antihypertensives (25.1%), non-steroidal anti-inflammatory drugs (24.5%), and anticoagulants/antithrombotics (19.5%). Most recent laboratory results revealed mean levels of hemoglobin, fetal hemoglobin, hematocrit, and platelets of 8.9±1.7 g/dL, 10.2±12.2%, 25.3±5.1%, and 347.4±151.4x109/L, respectively. Summary/Conclusion: The vast majority of SCD patients living in Portugal were under HU. There was a high incidence of documented acute and chronic complications, highlighting the clinical complexity these patients: nearly 90% had at least one comorbidity and 73% experienced pain episodes. An interesting data was the negative correlation between the number of VOC and the patient’s BMI, establishing the importance of weight control in SCD patients. This study provides the first real-world characterization of adult patients diagnosed with SCD living in Portugal. It contributes to a better understanding of the disease and provides important insights to improve patient management and care. Keywords: Sickle cell patient, Real world data, Epidemiology, Sickle cell disease

Gain-of-function mutations in the EPAS1/HIF2A gene have been identified in patients with hereditary erythrocytosis that can be associated with the development of paraganglioma, pheochromocytoma and somatostatinoma. In the present study, we describe a unique European collection of 41 patients and 28 relatives diagnosed with an erythrocytosis associated with a germline genetic variant in EPAS1. In addition we identified two infants with severe erythrocytosis associated with a mosaic mutation present in less than 2% of the blood, one of whom later developed a paraganglioma. The aim of this study was to determine the causal role of these genetic variants, to establish pathogenicity, and to identify potential candidates eligible for the new hypoxia-inducible factor-2 α (HIF-2α) inhibitor treatment. Pathogenicity was predicted with in silico tools and the impact of 13 HIF-2b variants has been studied by using canonical and real-time reporter luciferase assays. These functional assays consisted of a novel edited vector containing an expanded region of the erythropoietin promoter combined with distal regulatory elements which substantially enhanced the HIF-2α-dependent induction. Altogether, our studies allowed the classification of 11 mutations as pathogenic in 17 patients and 23 relatives. We described four new mutations (D525G, L526F, G527K, A530S) close to the key proline P531, which broadens the spectrum of mutations involved in erythrocytosis. Notably, we identified patients with only erythrocytosis associated with germline mutations A530S and Y532C previously identified at somatic state in tumors, thereby raising the complexity of the genotype/phenotype correlations. Altogether, this study allows accurate clinical follow-up of patients and opens the possibility of benefiting from HIF-2α inhibitor treatment, so far the only targeted treatment in hypoxia-related erythrocytosis disease.

Sickle cell disease is a hereditary multiorgan disease that is considered rare in the EU. In 2017, the Rare Diseases Plan was implemented within the EU and 24 European Reference Networks (ERNs) were created, including the ERN on Rare Haematological Diseases (ERN-EuroBloodNet), dedicated to rare haematological diseases. This EU initiative has made it possible to accentuate existing collaborations and create new ones. The project also made it possible to list all the needs of people with rare haematological diseases not yet covered health-care providers in the EU to allow optimised care of individuals with rare pathologies, including sickle cell disease. This Viewpoint is the result of joint work within 12 EU member states (ie, Belgium, Cyprus, Denmark, France, Germany, Greece, Ireland, Italy, Portugal, Spain, Sweden, and The Netherlands), all members of the ERN-EuroBloodNet. We describe the role of the ERN-EuroBloodNet to improve the overall approach to and the management of individuals with sickle cell disease in the EU through specific on the pooling of expertise, knowledge, and best practices; the development of training and education programmes; the strategy for systematic gathering and standardisation of clinical data; and its reuse in clinical research. Epidemiology and research strategies from ongoing implementation of the Rare Anaemia Disorders European Epidemiological Platform is depicted.

Hereditary erythrocytosis is a rare hematologic disorder characterized by an excess of red blood cell production. Here we describe a European collaborative study involving a collection of 2,160 patients with erythrocytosis sequenced in ten different laboratories. We focused our study on the EGLN1 gene and identified 39 germline missense variants including one gene deletion in 47 probands. EGLN1 encodes the PHD2 prolyl 4-hydroxylase, a major inhibitor of hypoxia-inducible factor. We performed a comprehensive study to evaluate the causal role of the identified PHD2 variants: (i) in silico studies of localization, conservation, and deleterious effects; (ii) analysis of hematologic parameters of carriers identified in the UK Biobank; (iii) functional studies of the protein activity and stability; and (iv) a comprehensive study of PHD2 splicing. Altogether, these studies allowed the classification of 16 pathogenic or likely pathogenic mutants in a total of 48 patients and relatives. The in silico studies extended to the variants described in the literature showed that a minority of PHD2 variants can be classified as pathogenic (36/96), without any differences from the variants of unknown significance regarding the severity of the developed disease (hematologic parameters and complications). Here, we demonstrated the great value of federating laboratories working on such rare disorders in order to implement the criteria required for genetic classification, a strategy that should be extended to all hereditary hematologic diseases.

| 2023; 7:S1 49 frequent painful crisis and extensive organ damage are common features of these patients. Pregnancy in SCD patients is associated with an increase in severe outcomes, namely, high risk of eclampsia and pre-eclampsia, stroke and even death. Therefore, it is crucial to maintain continuous medical surveillance during pregnancy, especially in women with previous strokes. Moreover, health services in lowand middle-income countries are generally not prepared to follow these patients. Aims: We aim to present the preliminary findings from the cohort study conducted at the Lucrecia Paim Maternity Hospital (Luanda, Angola), that aims to determine pregnancy complications in SCD women, especially those responsible for maternal death, and, by supporting the obstetric consultations in this hospital, contribute to the reduction of mortality and morbidity rates. Methods: Pregnancy monitoring includes analysis of clinical history and incidents (number of hospitalizations, blood transfusions, strokes and other clinical complications), hematological and biochemical analysis, transcranial doppler to assess cerebral hemodynamics and genetic analysis (confirmation of the diagnosis, genotyping of four SNPs in the β-cluster to assess the haplotype, and evaluation of the presence of the 3.7kb deletion of the α-globin gene). Results: To date, 61 women are being followed in the obstetric consultations, with ages from 18 to 40 years old (mean 26.1±5.4). There are no records of previous strokes, although 83.9% of the patients have been previously transfused (47 out of 56), 98.2% have been hospitalized (55 out of 56) due to SCD complications and 19.6% (10 out of 54) had at least one miscarriage. At the first appointment, total hemoglobin values ranged from 4.70 to 10.40 g/dL (n=52, mean 7.18±1.30), erythrocytes from 1.46 to 5.42 x1012/L (n=52, mean 2.46±0,72), white blood cells count from 1.67 to 61.88 x109/L (n=51, mean 12.20±8.69), platelets from 24.2 to 710.0 x109/L (n=52, mean 272.2±155.9), and lactate dehydrogenase (LDH) from 263.3 to 2836.7 (n=50, mean 708.1±450.46). The CAR/CAR haplotype, which is usually associated with a more severe prognosis, is the most prevalent in this population (57.7%, 30 out of 52), followed by the CAR/SEN haplotype (25.0%, 13 out of 52). In this population, 17.3% (9 out of 52) are homozygous for the 3.7kb α-thalassemia deletion and 44.2% (23 out of 52) are carriers. This deletion influences hematological and clinical aspects of sickle cell disease patients, resulting in less severe phenotypes. TCD time-averaged mean of the maximum velocity (TAMMx) at the middle cerebral arteries ranged between 41 to 132 cm/s (n=61, mean 84cm/s) and peak systolic velocity (PSV) from 61 to 180 cm/s (mean 129 cm/s). At the basilar artery level, TAMMx obtained were between 29 to 102 cm/s (n=60, mean 52 cm/s) and PSV ranged from 43 to 141 cm/s (mean 78 cm/s). Summary Conclusion: The main goal of this project is to study pregnancy-related complications and outcomes by giving support to an Angolan cohort of SCD pregnant women. We also intend to obtain TCD reference values of cerebral blood flow velocities in pregnant women with SCD as a risk predictor of vascular events as there are no values in the literature for this specific population.

Background Whole genome sequencing is increasingly being used for the diagnosis of patients with rare diseases. However, the diagnostic yields of many studies, particularly those conducted in a healthcare setting, are often disappointingly low, at 25–30%. This is in part because although entire genomes are sequenced, analysis is often confined to in silico gene panels or coding regions of the genome. Methods We undertook WGS on a cohort of 122 unrelated rare disease patients and their relatives (300 genomes) who had been pre-screened by gene panels or arrays. Patients were recruited from a broad spectrum of clinical specialties. We applied a bioinformatics pipeline that would allow comprehensive analysis of all variant types. We combined established bioinformatics tools for phenotypic and genomic analysis with our novel algorithms (SVRare, ALTSPLICE and GREEN-DB) to detect and annotate structural, splice site and non-coding variants. Results Our diagnostic yield was 43/122 cases (35%), although 47/122 cases (39%) were considered solved when considering novel candidate genes with supporting functional data into account. Structural, splice site and deep intronic variants contributed to 20/47 (43%) of our solved cases. Five genes that are novel, or were novel at the time of discovery, were identified, whilst a further three genes are putative novel disease genes with evidence of causality. We identified variants of uncertain significance in a further fourteen candidate genes. The phenotypic spectrum associated with RMND1 was expanded to include polymicrogyria. Two patients with secondary findings in FBN1 and KCNQ1 were confirmed to have previously unidentified Marfan and long QT syndromes, respectively, and were referred for further clinical interventions. Clinical diagnoses were changed in six patients and treatment adjustments made for eight individuals, which for five patients was considered life-saving. Conclusions Genome sequencing is increasingly being considered as a first-line genetic test in routine clinical settings and can make a substantial contribution to rapidly identifying a causal aetiology for many patients, shortening their diagnostic odyssey. We have demonstrated that structural, splice site and intronic variants make a significant contribution to diagnostic yield and that comprehensive analysis of the entire genome is essential to maximise the value of clinical genome sequencing.