Approximately 3500 children with sickle cell disease (SCD) are born in Brazil each year, but the burden of SCD morbidity is not fully characterised. A large, multi-centre cohort was established to characterise clinical outcomes in the Brazilian SCD population and create the infrastructure to perform genotype-phenotype association studies. Eligible patients were randomly selected from participating sites and recruited at routine visits. A biorepository of blood samples was created and comprehensive demographic and clinical outcome data were entered in a centralized electronic database. Peripheral blood genome-wide single nucleotide polymorphism (SNP) genotyping was performed using a customized Transfusion Medicine (TM) Array. A total of 2795 participants at six Brazilian sites were enrolled between 2013 and 2015. The cohort included slight predominance of children <18 years (55·9%) and females (53·0%). Haemoglobin (Hb) SS was the most common SCD genotype (70·7%), followed by HbSC (23%), Sβ0 (3·0%) and Sβ+ (2·9%). SNP data from the TM Array were analysed to evaluate the genetic ancestry of the cohort and revealed significant admixture among the population. Demographics and clinical complications, stratified by age and SCD genotype, are summarized and future studies in this cohort are discussed.

There is a high incidence of stroke in male children and in children with SCA. Coexistence with α-thal and haplotypes of the beta globin chain cluster did not show any significant association with stroke. The heterogeneity between previously evaluated populations, the non-reproducibility between studies, and the need to identify factors associated with stroke in patients with SCA indicate the necessity of conducting further research to demonstrate the relevance of genetic factors in stroke related to SCD.

BACKGROUNDRed blood cell (RBC) transfusions are used in sickle cell disease (SCD) to treat acute complications or as chronic transfusion therapy (CTT) to prevent severe manifestations. The objectives of this study were to describe blood utilization and adverse events (AEs) associated with RBCs in the Brazilian SCD population and compare characteristics of patients treated or not with CTT.STUDY DESIGN AND METHODSA SCD cohort was established at six Brazilian centers. Medical and blood bank records were abstracted for clinical and transfusion history. Two controls not treated with CTT matched on center, SCD genotype, sex, and age were selected for each CTT case within the cohort to compare characteristics between the two groups.RESULTSMost of the 2794‐member cohort had received a transfusion (75.0% of children and 89.2% of adults) with 29.2% of patients receiving transfusion in the prior year. There were 170 (10.6%) children and 115 (9.2%) adults treated with CTT. Children not treated with CTT were more likely to have pain and acute chest hospitalizations in the prior year (25.3% vs. 11.9%, p = 0.0003; and 22.0% vs. 10.7%, p = 0.002, respectively). Both iron overload and alloimmunization were more common in CTT cases compared to controls (65.6% vs. 17.0% and 36.2% vs. 15.9%, respectively). A higher proportion of adults treated with CTT demonstrated oxygen saturation of greater than 95% compared to controls not treated (51.1% vs. 39.2%), while there was no difference in oxygenation between children treated or not. Of 4501 transfusion episodes, 28 (0.62%) AEs were reported. There was no difference in AEs associated with transfusions for acute indications versus CTT.CONCLUSIONRed blood cell transfusion was common in Brazilian SCD patients, with utilization driven by CTT. Transfusion reactions were not common; however, alloimmunization and iron overload were frequent among those on CTT, highlighting the need for novel clinical strategies to mitigate these risks.

Introduction Priapism is the persistent and painful erection of the penis and is a common sickle cell disease (SCD) complication. Aim The goal of this study was to characterize clinical and genetic factors associated with priapism within a large multi-center SCD cohort in Brazil. Methods Cases with priapism were compared to SCD type-matched controls within defined age strata to identify clinical outcomes associated with priapism. Whole blood single nucleotide polymorphism genotyping was performed using a customized array, and a genome-wide association study (GWAS) was conducted to identify single nucleotide polymorphisms associated with priapism. Main Outcome Measure Of the 1,314 male patients in the cohort, 188 experienced priapism (14.3%). Results Priapism was more common among older patients (P = .006) and more severe SCD genotypes such as homozygous SS (P &lt; .0001). In the genotype- and age-matched analyses, associations with priapism were found for pulmonary hypertension (P = .05) and avascular necrosis (P = .01). The GWAS suggested replication of a previously reported candidate gene association of priapism for the gene transforming growth factor beta receptor 3 (TGFBR3) (P = 2 × 10−4). Clinical Implications Older patients with more severe genotypes are at higher risk of priapism, and there is a lack of consensus on standard treatment strategies for priapism in SCD. Strengths & Limitations This study characterizes SCD patients with any history of priapism from a large multi-center cohort. Replication of the GWAS in an independent cohort is required to validate the results. Conclusion These findings extend the understanding of risk factors associated with priapism in SCD and identify genetic markers to be investigated in future studies to further elucidate priapism pathophysiology.

Sickle cell disease (SCD) is associated with significant morbidity, and allogeneic hematopoietic stem cell transplantation (HSCT) remains the primary curative treatment. Recently, the Brazilian Ministry of Health released a regulation that required the publically funded healthcare system to pay for HSCT for SCD patients with defined indications. We used an existing 2794-member SCD cohort established during 2013 to 2015 to characterize candidates for HSCT and estimate the number of possible donors. Of 2064 patients with SC anemia (SCA), 152 of 974 children (16%) and 279 of 1090 adults (26%) had at least 1 HSCT indication. The most common indication for transplant was stroke (n = 239) followed by avascular necrosis (n = 96), priapism (n = 82), cerebrovascular disease (n = 55), >2 vaso-occlusive episodes (n = 38), alloantibodies and chronic transfusion therapy (n = 18), and >2 acute chest syndrome episodes (n = 11). Increasing age, number of transfusions, abnormal transcranial Doppler, retinopathy, dactylitis, and use of hydroxyurea were more frequent in the 152 children with an indication for HSCT compared with 822 without (P < .001). Of 152 children and 279 adults meeting the eligibility definition, 77 (50%) and 204 (73%), respectively, had at least 1 non-SCD full sibling who could potentially serve as a donor. In conclusion, in a large cohort of SCA patients, 16% of children and 26% of adults had at least 1 indication for HSCT; these indications were associated with the severity of the disease. This study provides clinical data necessary for estimating the costs and infrastructure that would be required to implement HSCT in a public healthcare system.

BackgroundThe diagnosis of sickle cell disease (SCD) is made by hemoglobin assays such as high-performance liquid chromatography (HPLC), isoelectric focusing and cellulose acetate or citrate agar electrophoresis. These assays are easy to perform and used in large-scale newborn screening in many countries. These tests however may not easily differentiate Sβ0 thalassemia from SS or identify other hemoglobin variants, and in this case, hemoglobin (HBB) gene sequencing may be necessary.ObjectivesTo develop a high throughput DNA based confirmatory assay for SCD and to detect mutations in the HBB geneMethodsWe developed an automated pyrosequencing technique (PyS) based on QIAGEN technology (Hilden, Germany) to detect homozygous or heterozygous hemoglobin S mutations as well as hemoglobin C mutations. The technique was tested on 2,748 samples from patients enrolled in a multi-center SCD cohort in Brazil. Patients were previously tested using HPLC to diagnose SCD as part of routine clinical care. Any subjects with discrepant results between HPLC and PyS or with heterozygous hemoglobin S detected had Sanger sequencing of the HBB gene.ResultsWe identified 168 samples with discrepant results between HPLC and PyS and 100 with concordant PyS = heterozygous S and HPLC, which would suggest SB-thalassemia or other heterozygous S variants. The PyS assay correctly identified 1906 (98.7%) of the 1930 HbSS and 628 (98.7%) of the 636 HbSC samples. Of the 179 remaining samples, PyS correctly indicated S heterozygosis in 165 (92.2%). Of the 165 heterozygous S samples confirmed by Sanger as consistent with Sβ thalassemia genotype, 84 samples were classified as Sβ0 thalassemia and 81 as Sβ+ thalassemia. The most frequent beta thalassemia mutations of Sβ0 and Sβ+ were HBB: c.118C>T (Gln40Stop) and HBB c.92 + 6T> C, respectively.DiscussionThe PyS proved to be satisfactory for large-scale confirmatory testing of hemoglobin mutation. Moreover, with this study we were able to describe the most common β+ and β0 mutations in SCD patients with Sβ-thalassemia in a large multi-institutional SCD cohort in Brazil.

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22Background: The diagnosis of sickle cell disease (SCD) is made by hemoglobin assays such 23 as high-performance liquid chromatography (HPLC), isoelectric focusing and cellulose 24 acetate or citrate agar electrophoresis. These assays are easy to perform and used in large-25 scale newborn screening in many countries. These tests however may not easily differentiate 26 Sβ 0 thalassemia from SS or identify other hemoglobin variants, and in this case, hemoglobin 27 (HBB) gene sequencing may be necessary. Objectives: To develop a high throughput DNA 28 based confirmatory assay for SCD and to detect mutations in the HBB gene. Methods: We 29 developed an automated pyrosequencing technique (PyS) based on QIAGEN technology 30 (Hilden, Germany) to detect homozygous or heterozygous hemoglobin S mutations as well as 31 hemoglobin C mutations. The technique was tested on 2,748 samples from patients enrolled in 32 a multi-center SCD cohort in Brazil. Patients were previously tested using HPLC to diagnose 33 SCD as part of routine clinical care. Any subjects with discrepant results between HPLC and 34 PyS or with heterozygous hemoglobin S detected had Sanger sequencing of the HBB gene.35 Results: We identified 168 samples with discrepant results between HPLC and PyS and 100 36 with concordant HPLC and PyS= heterozygous S, which would suggest Sβ-thalassemia or 37 other hemoglobin S variants. The PyS assay correctly identified 1906 (98.7%) of the 1930 38 HbSS and 628 (98.7%) of the 636 HbSC samples. Of the 179 remaining samples, PyS 39 correctly indicated S heterozygosis in 165 (92.2%). Of the 165 heterozygous S samples 40 confirmed by Sanger as consistent with Sβ thalassemia genotype, 84 samples were classified 41 as Sβ 0 thalassemia and 81 as Sβ + thalassemia. The most frequent beta thalassemia mutations 42 of Sβ 0 and Sβ + were HBB: c.118C>T (Gln40Stop) and HBB c.92 + 6T> C, respectively.43Discussion: The PyS proved to be satisfactory for large-scale confirmatory testing of 44 hemoglobin mutation. Moreover, with this study we were able to describe the most common 45 3 β + and β 0 mutations in SCD patients with Sβ-thalassemia in a large multi-institutional SCD 46 cohort in Brazil. 47