ALBERT

Description: Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a disorder due to an acquired loss-of-function mutation in the phosphatidylinositol glycan class A (PIG-A) gene. A large spectrum of acquired PIG-A mutations has been described, like insertions or deletions involving a single base or several bases, and single base substitution that are the most common. Usually there are more than one PIGA mutations and one clone is predominant. The clinical manifestations of PNH are intrinsically related to clonal expansion of hematopoietic stem cell deficient in GPI-anchored proteins. Some hypothesis failed to explain alone this clonal expansion. Here we try to identify and to correlate PIG-A gene mutations with clinical manifestations in a series of patients with PNH. Methods: We analyzed 31 patients with classical PNH (n=23) or aplastic anemia and PNH clone (n=8). The sequencing of the PIG-A gene was performed using the Sanger technique. The electropherograms were aligned against the reference sequence of the PIG-A gene deposited in GenBank (Accession number NG_009786), and analyzed using the Geneious R10 software (Biomatters). After analysis, a search was performed in the Clinvar, dbSNP and HGMD databases to verify the pathogenicity of the mutations. For variants without description in the literature, a pathogenicity prediction analysis was performed using Mutation Taster, Polyphen 2 and Human Splicing Finder software. Results: We found 29 different variants of the PIG-A gene in 27 patients: 23 were new mutations, with no previous description in the literature, 3 were previously described mutations, and 3 were single nucleotide polymorphism (SNP). There was great variation in the type and location of somatic mutations. Mutations were predominantly small deletions and simple base changes; 42% of the mutations were described as frameshift mutations and 31% missense mutations. We did not find any specific correlation between the clinical characteristics of hemolytic PNH patients and their mutations, due to the wide variety of mutations. According the pathogenicity prediction programs, the majority (22 of 29) of the variants found were classified as probably pathogenic. Among the 23 patients with hemolytic PNH, 19 patients had at least one mutation classified as pathogenic. In patients with subclinical PNH, only SNPs were found. Fifteen patients with hemolytic PNH had more than one concomitant mutation, most of which were probably pathogenic mutations associated with a polymorphism. Conclusion: We described PIG-A mutations in a series of PNH patients in Brazil and observed no correlation exists between mutation types and clinical features in hemolytic patients. Among subclinical PNH patients, only SNPs were observed, probably because of small clone sizes. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a nonmalignant clonal disease of hematopoietic cells due to acquired mutations in the phosphatidylinositol glycan class A (PIG-A) gene, which is required for glycosylphosphatidylinositol (GPI) anchor biosynthesis. This leads to partial or complete absence of all GPI-linked proteins, who are complement regulatory proteins, resulting in an increased sensitivity of the red blood cells to the action of complement. PNH is characterized by signs and symptoms related to intravascular hemolysis, hypercoagulability state, and varying degrees of medullary insufficiency. The anti-complement therapy radically changed the PNH patients outcomes. However, there are little data on the clinical characteristics of PNH in Latin American countries. Methods: We performed a retrospective analysis of 109 patients with PNH clone followed from January 1987 until July 2019 in two Brazilian centers: Universidade Federal de Sao Paulo and Hospital Sirio Libanes (Sao Paulo-Brazil). Most patients (88%) were evaluated while the others had lost follow up or died and data was obtained from their medical reports. Patients were separated into 3 groups: classical PNH (n=44) PNH associated with other bone marrow disorder(n=12), and subclinical PNH, defined as PNH clone (at least 0.01% of cells with PNH clone) associated with another bone marrow disorder (n=53, aplastic anemia in 95% of cases). Median follow up was 60 months (range: 3-394). Results: Median age at diagnosis was 41 years (range: 18-81), and 51% were male. Among the 56 patients with hemolytic PNH, 86% had fatigue, 66% hemoglobinuria, 45% abdominal pain and 16% dysphagia. Venous thromboembolism was observed in 14 cases (25%), with abdominal thrombosis in 7 cases (50%). Seven patients (13%) had arterial thrombosis (stroke or transient ischemic attack). Only 5 patients (10%) in the hemolytic group had acute renal failure and needed dialysis therapy due to a hemolytic crisis, but progressed to recovery of renal function after the event. No patient in this series had moderate or severe chronic kidney disease. Most hemolytic patients (73%) were treated with eculizumab, with a median time from diagnosis to the start of eculizumab of 25 months (range: 2-275). All eculizumab-treated patients had significant reduction in intravascular hemolysis with lactate dehydrogenase (LDH) normalization. Most had significant improvement in anemia, with increase in the median hemoglobin from 9.1 g/dL before treatment to 11.7 g/dL after eculizumab. The vast majority (94%) became transfusion-independent. Overall survival (OS) at 5 years was 100% at 5 years for classical PNH (n=44), 89% for subclinical PNH (n=53) and 71% for PNH associated with another bone marrow disease (n=12). Conclusion: The clinical data and the distribution of the three subtypes of PNH in this study in this large series of Brazilian PNH patients were similar to other published series, except for a lower frequency of venous or arterial thrombosis in hemolytic patients before eculizumab treatment and a lower frequency of chronic kidney disease in our series. We also confirmed in our series the efficacy of eculizumab in controlling hemolysis and PNH-related complications and death risk. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 5146 INTRODUTION: Most hereditary hemochromatosis (HH) patients are homozygous for the p.C282Y mutation in the HFE gene. But rare HFE variants have been shown to be associated with HH. In addition, four main types of non-HFE HH are caused by mutations in the hemojuvelin (HJV), hepcidin (HAMP), transferrin receptor 2 (TFR2) and ferroportin (SLC40A1) genes. The main aim of this study was to screen for HFE, HJV, HAMP, TFR2 and SLC40A1 mutations and to investigate their relationship with HH. MATERIAL E METHODS: Fifty-one Brazilian patients with primary iron overload (transferrin saturation 〉 50% in females and 60% in males) were eligible. Subsequent bidirectional sequencing for each exon of HFE, HJV, HAMP, TFR2 and SLC40A1 genes was performed. The effect of HFE p.V256I novel mutations on protein structure was analyzed by in silico molecular dynamic and free energy calculations. RESULTS: Thirty-seven (72.5%) out of the 51 patients presented at least one HFE mutation. The most frequent genotype associated with HH was the homozygous p.C282Y mutation (n = 11, 21.6%). In addition, the p.S65C mutation was found in heterozygosis with p.H63D in two patients and the homozygous genotype for the p.H63D was found in two patients. One novel mutation (p.V256I) was indentified in heterozygosis with the p.H63D mutation. In silico modeling analysis of protein behavior suggested that the p.V256I mutation does not reduce the binding affinity between HFE and β2-microglobulin (β2M) in the same way the p.C282Y mutation does compared with the native HFE protein. Sequencing HJV revealed one patient presenting Juvenile hemochromatosis (JH) (homozygous genotype for the HJV p.G320V mutation); two patients carrying heterozygous genotype for the p.E302K mutation; and one patient with heterozygosis p.A310G polymorphism. Sequencing HAMP revealed one patient carrying p.P48G novel mutation in the heterozygous form. Three and five non-pathogenic polymorphisms were observed in the TFR2 and SLC40A1 genes, respectively. Sequencing SLC40A1 also identified one patient with homozygous genotype for the p.R561G described mutation; and one patient with homozygous genotype for the p.G204S novel mutation. CONCLUSION: The HFE p.C282Y in homozygosis or in heterozygosis with p.H63D was the most frequent mutation associated with HH in our sample population. The novel HFE p.V256I mutation could not be implicated in the molecular basis of the HH phenotype. Two described mutations, HJV p.E302K and SLC40A1 p.R561G, could have functional consequences according to previously studies contributing to HH phenotype. Two novel mutations, HAMP p.R48G and SLC40A1 p.G204S, may be implicated with iron overload in these patients, but further studies are need to explain their impact on proteins. Disclosures: No relevant conflicts of interest to declare.

Description: Background In Brazil, wheat and corn flour is fortified with 150 µg of folic acid (FA), the synthetic form of folate. Individuals with increased cell duplication, including pregnant women and patients with hemolytic anemia need increased amounts of folate. The effects of amounts of FA higher than the defined tolerable upper intake of 1 mg/day are poorly understood. Some Brazilian patients with hemolytic anemia, such as hereditary spherocytosis (HS), have been receiving 5mg/day supplemental FA, in addition to being exposed to mandated food fortification with FA. Our previous data has shown that patients with HS have higher serum folate levels than healthy controls, as well as higher mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 genes (1). However, it was not clear whether the increased mRNA expression resulted from folic acid use or underlying disease. Objective The aim of this study was to verify the effects of an intervention with 5mg/day FA on folate levels (serum and whole blood), serum inflammatory markers levels, mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 genes and cytotoxicity of NK cells in healthy Brazilian volunteers. Material and methods Fifteen male and fifteen female healthy subjects were given 5mg/day FA for 90 days. Blood was collected at baseline, day 45 and day 90 for blood count, including reticulocytes, C-reactive protein and lactate dehydrogenase (LDH). Folate (serum and whole blood) and vitamin B12 were determined by a microbiological method. Serum cytokines levels were measured using a Milliplex Map kit. The mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 genes in mononuclear cells were performed using Real Time PCR. Cytotoxicity of lymphocytes and NK cell number were measured by flow cytometry. Results All blood count parameters were unaffected by FA intervention, whereas there was a slight increase in concentrations of LDH (P = 0.001) after 90 days compared with baseline and 45 day measurements. The folate levels (serum and whole blood) were higher at 45 and 90 days of intervention with 5mg/day of FA (P0.05). The mRNA expression of IL8 was higher at 45 days of intervention (Fig 1), while mRNA expressions of TNF-α were elevated at 45 and 90 days compared with baseline (Fig 1). No difference was found in mRNA of DHFR, MTHFR and IFNγ in this study. The data are median and interquartile intervals. The groups were compared using the Friedman test, when significant was performed for multiple comparisons Dunn`s test. Different letters show significant differences between groups After 5 mg FA daily there was a reduction in the number and cytotoxic capacity of NK cells (Table 1). The data are median and interquartile intervals. The groups were compared using the Friedman test, when significant was performed for multiple comparisons Dunn`s test. Different letters show significant differences between groups. Conclusions Intervention with 5 mg/day of FA in healthy people was associated with around 4-fold increase in serum and whole blood folate, accompanied by increased mRNA expression of proinflammatory cytokines IL8 and TNF-α and a reduction in NK cell number and cytotoxicity. High dose FA fortification may result in changes in innate immune parameters that could perturb immune surveillance pathways. Financing: FAPESP 2012/12912-1, CNPq 4826412012-6 and CNPq 401586/2014-6 References 1. Paniz, C et al. Blood 2014;124:4005. Presented at the ASH 2014. Figure 1. mRNA expression of IL8 (A) and TNFα (B) genes in healthy subjects before and 45 and 90 days after 5 mg FA daily Figure 1. mRNA expression of IL8 (A) and TNFα (B) genes in healthy subjects before and 45 and 90 days after 5 mg FA daily Tabel 1 Number, lytic activity and cytotoxic capacity of NK cells after intervention with 5 mg/day of folic acid Tabel 1. Number, lytic activity and cytotoxic capacity of NK cells after intervention with 5 mg/day of folic acid Disclosures No relevant conflicts of interest to declare.

Description: BACKGROUND: Sickle Cell Anemia (SCA) presents extra and intravascular hemolysis. The eryptosis depends on phagocytosis signs like CD47, which is known as a "don't eat me" signal. Other mechanisms associated to hemolysis, such as complement activation mediated by CD59, are poorly understood. One of the effects of hydroxycarbamide (HU) is the reduction of hemolysis, but little is known about the action of HU in these mechanisms. OBJECTIVES: To evaluate markers related to hemolysis in patients with SCA at steady state, with and without HU. METHODS: We studied 40 adult patients with SCA (n=36) or Sβ0-Thalassemia (n=4), 70% females, median age 30 years (range 18-70y). The patients were followed at the Outpatient Clinic of EPM /UNIFESP. Inclusion criteria: patients in use of HU in maximum tolerated dose for more than 1 year (G1, n=20); or without HU (G2, n=20). Exclusion criteria: pregnant women and chronic transfusion. Laboratory evaluation: CBC, reticulocyte (Ret), fetal hemoglobin (HbF), indirect bilirubin, lactate dehydrogenase (LDH), LDH isoforms, free Hb (free-Hb), haptoglobin (Hp), hemopexin, phosphatidylserine, microvesicles (Mcv), Howell-Jolly bodies (in erythrocytes and Ret), and expression of band-3, CD47 (in erythrocyte and Ret) and CD59 (in erythrocytes and Mcv). The last parameters were evaluated by flow cytometry (FACS Calibur®, BDB, San Jose, CA), using specific antibodies according to the manufacturer instructions. Data were analyzed by CellQuest® software (BDB), and the results expressed in median fluorescence intensity or percentage of positivity. Statistical analysis: Mann-Whitney test and Pearson's correlation, with significance level of 5%. RESULTS: Themean corpuscular volume and HbF were higher in G1 than G2 (p=0.033, p=0.0001, respectively) (Table 1). Free-Hb, LDH and isoforms LDH-1, -2, and -3 were lower in G1 (p=0.023, p=0.003, p=0.005, p=0.001, p=0.002, respectively). There were correlation between free-Hb and LDH-1 (r=0.45, p=0.003), LDH-2 (r=0.80, p=0.001), and LDH-3 (r=0.71, p=0.001). As expected, Hp was diminished in all patients, however G1 patients showed higher values (p=0.040). The expression of band 3 and of CD59, in erythrocytes and Mcv, were higher in G1 (p=0.001, p

Description: Background: Sickle cell anemia (SCA) is a complex disease, associated with hemolysis, vaso-occlusion, vascular inflammation and endothelial activation. Significant morbidity and premature mortality are hallmarks of the disease and elevation of tricuspide regurgitant velocity (TRV), determined by doppler-echocardiography, has been associated with higher risk. The identification of biomarkers associated with severity in SCA is desirable. Circulating serum microRNAs (miRNA) are molecular targets studied in different diseases as diagnostic or prognostic markers, however there are few studies in SCA. Purpose: to identify specific signatures of miRNAs in plasma samples of SCA patients according to severity indexes. Methods: Screening of the miRNAs expression was performed in 8 patients. These patients were classified by TRV measure (referred as TRV Score): 4 samples with TVR ≥ 2.5 m/s and 4 with TRV 〈 2.5 m/s. After extraction of total RNA, the samples were analyzed by real-time PCR using Megaplex RT Human Pool A and Megaplex RTHuman Pool Blife (Thermofisher) comprising 667 distinct miRNAs. Expression Suite Software (Thermofisher) and SPSS were used for analysis. Seventeen miRNAs were differentially expressed between the two groups (p 〈 0.05) and miR16 was considered as the endogenous candidate. Five differentially expressed miRNAs (miR15b, miR502, miR510, miR544, miR629) were selected for validation in 56 patient samples using TRV Score. Another two severity scores were also used: (a) Organ Injury Score (SLO) - based on the presence or absence of 5 lesions: stroke, TRV ≥ 2.5 m/s, leg ulcers, osteonecrosis, and microalbuminuria (adapted from Afenyi-Annan et al. Transfusion 2008; 48:197) and (b) NIH Bayesian score - available online (http://bios.ugr.es/dss-calculator/). The ROC curve was used to analyze the data of relative expression (2-ΔCT) of each miRNA. Results: Two out of five miRNA, miR510 and miR629, were significantly decreased in more severe patients. The miR510 expression allowed the discrimination of the patients according to TRV Score at the cutoff 0.000331043, sensitivity 71.4%, specificity 73.3% and AUC of 0.825 (95% CI: 0.689-0.962, p = 0.001). The same miRNA was also a good discriminant in the SLO at a cutoff of 0.000331043, sensitivity 77.8%, specificity 72.2% and area under the curve (AUC) of 0.769 (95% CI: 0.666-0.931), p = 0.008. The miR629 was related to severity according to the Bayesian Score at the cutoff of 0.0009854449, sensitivity 66.7%, specificity 75% and AUC of 0.729 (95% CI: 0.552-0.907, p = 0.027). The other miRNAs, miR15b, miR502 and miR544, showed no significant results. Discussion and Conclusions: This is the first study which looks into plasma miRNA as a biomarker of SCA severity. The miR510 regulate Peroxirredoxin-1 (PRDX1), a protein involved in the stress-oxidation. Increased oxidative stress presented in SCA might imply that miR510 has a role in this mechanism. The miR629 appears to be involved in the AKT1 signaling pathway related to Endothelin-1. As it is known, endothelin-1 is increased in SCA and is important in pulmonary hypertension. The miR510 and miR629 appear to be hypoexpressed in patients with more severe SCA, probably with greater importance in the regulation of clinical manifestations associated with vascular disease, although more studies seem to be necessary. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4843 Periodic limb movements during sleep (PLMS) are commonly described in children with Sickle Cell Disease (SCD), however the relevance of such events in sleep disturbance or complaint of daytime fatigue remain questionable. It has been known that the dopaminergic neurotransmission has a modulator role in pain perception. Since PLMS have been suggested to be a potential biological marker of dopaminergic mechanisms, this study aimed to assess PLMS in adults with SCD and its correlations with clinical and sleep parameters. Methods: Seventy adults with SCD (50% females, matched for age and body mass index), underwent Brief Pain Inventory, overnight polysomnogram and laboratorial tests for hemoglobin, reticulocytes, ferritin, transferin saturation, haptoglobin, fetal hemoglobin, lactate dehydrogenase (LDH), and bilirrubins. Results: The mean PLMS index was higher in females (16.5±10.7/h vs. 8.7±8.2/h, p〈 0.05), with 88.6% of the females having increased PLMS index (≥ 15/h) in comparison to males (22.6), p

Description: The mutation causing sickle cell anemia (rs334, GAG-GTG, glu6val) had several independent origins in Africa, the Middle East and India and spread throughout parts of the world by wars, slave trading and population migrations. The genetic background upon which the HbS mutation occurred, or the β-globin gene (HBB) haplotype, is associated with differences in the phenotype of this disease and the ability of affected individuals to synthesize fetal hemoglobin (HbF). The main modifier of the disease phenotype is the level of HbF in the blood of affected individuals. HbF inhibits the polymerization of HbS, the proximate cause of disease pathophysiology. As part of the NHLBI NextGen consortium (U01HL107443) we established a library of induced pluripotent stem cells (iPSC) from patients with sickle cell anemia of diverse HBB haplotypes and HbF phenotypes. The purpose of establishing this library was to allow genetic studies of globin gene expression during the erythroid differentiation of iPSC of diverse genotypes. During these studies we have implemented an efficient and highly reproducible platform for the production of large numbers of sickle cell anemia-specific iPSC, derived and characterized a novel in vitro system for the production of an unlimited supply of erythroid lineage cells from the directed differentiation of normal and disease-specific iPSC and used this system to recapitulate erythroid-lineage ontogeny in vitro with the sequential development of primitive and definitive erythropoiesis, accompanied by the appropriate expression of stage-specific globin genes. We have recently finished whole genome DNA and RNA sequencing analysis in some of these lines aimed at identifying developmental gene expression profile differences between erythroid precursors that produce primarily HbF and those that produce primarily HbA or HbS as part of our search for novel HbF genetic modifiers associated with markedly elevated HbF levels found in sickle cell anemia patients naturally, or in response to hydroxyurea treatment. Furthermore, our labs are also focusing on using a CRISPR-based gene editing platform to study the effect of novel HbF genetic modifiers and explore globin switching. Cell lines established are shown in the table. Table 1. Number of subjects recruited to date 98 Number of subjects with iPSC lines established 56 Average number of iPSC lines per subject 3 (total of 158 lines generated) Quality control status of iPSC lines All lines are expanded and banked, mycoplasma free, express pluripotency markers Subjects with target cells differentiated (erythrocytes) 25 Samples have been collected on African American patients with sickle cell anemia with diverse HBB haplotypes, predominantly homozygotes and compound heterozygotes for the Benin and Bantu haplotypes, Saudi Arabian patients with the Arab-Indian haplotype and the Saudi Benin haplotype that is characterized by HbF levels about twice as high as in African Benin haplotype patients and from Brazilian patients who are predominantly homozygotes for the Bantu haplotype that typically is associated with the lowest HbF of all HBB haplotypes. This iPSC-based library and the data associated with it represents a valuable readily available resource for the sickle cell research community and all the generated lines will be available for distribution early in 2016 through WiCell. Disclosures No relevant conflicts of interest to declare.