Initial Genomic Analysis of a Pure Erythroid Leukemia Developing in Association with Hydroyurea Treatment for Sickle Cell Anemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3254-3254
Author(s):  
Zhengyuan Wang ◽  
Deepika S. Darbari ◽  
J. Joseph Melenhorst ◽  
Zachariah A. McIver ◽  
Irina Maric ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Conflicts Of Interest ◽  
Sequence Data ◽  
Genomic Analysis ◽  
Stress Erythropoiesis ◽  
Chromosome 19P ◽  
Mosaic Aneuploidy ◽  
Tumor Dna ◽  
Exome Sequence

Abstract Abstract 3254 The antimetabolite hydroxyurea (HU) is the only widely available therapy for reducing complications of sickle cell anemia. It is not widely prescribed due to concerns about adverse effects, including cancer. Insufficient data currently exist regarding its leukemogenic potential. HU associated leukemia in sickle cell anemia is exceptionally rare, with only 6 literature reports. Whether such cases are coincidental or related to HU remains unanswered. We identified a 33 year old male with pure erythroid leukemia (formerly M6 AML) and a unique combination of sickle cell anemia, HU treatment, and short treatment latency. Cytogenetics showed a complex karyotype with 5q rearrangement, 7q deletion, losses of chromosomes 15–22 and Y, suggesting a therapy related etiology. To date, no sickle cell anemia AML cases have undergone in-depth genomic analysis to determine if HU has an etiologic role in susceptibility to therapy related leukemogenesis. High density SNP genotyping was performed with 2.5 million markers to understand progressive chromosomal disease changes. Comparison of germline DNA prior to HU and tumor DNA identified 10 mosaic aneuploidy events present at AML diagnosis including losses at 5q, 7q and 17p and gains at 19p. Whole exome sequencing was also performed on the patient's germline, tumor and relapsed tumor DNA. We identified 33 putative somatic missense mutations including a validated somatic TP53 mutation, however somatic mutations were not present in commonly mutated AML genes like NPM1, RUNX1 or IDH1. Genome-wide cytogenetic band enrichment analysis of exome sequence data using the Gene Set Analysis Toolkit suggests the presence of a significant enrichment of mutations along chromosome 19p, and the same analysis of peripheral blood gene expression from an unrelated cohort of 10 sickle cell anemia subjects with brisk hematopoietic activity also suggested association of chromosome 19p with stress erythropoiesis. Together, the mosaic aneuploidy and exome sequence analysis converged on a region of 19p potentially important to the pathogenesis of pure erythroid leukemia and possibly stress erythropoiesis. This region extends at least 1.9 Mb and includes 58 genes. We are validating mutations in this region and exploring the role of 19p during stress erythropoiesis to further determine the significance of this finding. Specifically, several 19p mutations have been introduced into the BaF3 cell line to investigate their role in myeloid cell growth. Investigation of this rare leukemia case offers a unique opportunity to elucidate the pathogenesis of erythroleukemia and possibly to determine if HU therapy is associated with leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

Decrease in Microvascular Blood Flow in Sickle Cell Anemia Is Triggered by Autonomic Signals and Not Directly by Hypoxia: A New Hypothesis for Sickle Crisis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1523-1523
Author(s):  
Suvimol Sangkatumvong ◽  
Roberta Kato ◽  
Jon A Detterich ◽  
Herbert J Meiselman ◽  
John C Wood ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Conflicts Of Interest ◽  
Genetic Disorder ◽  
Arterial Oxygen Saturation ◽  
Substantial Reduction ◽  
Microvascular Blood Flow ◽  
Arterial Oxygen ◽  
Experimental Session ◽  
Peripheral Vasoconstriction

Abstract Abstract 1523 Poster Board I-546 Sickle cell anemia (SCA) is a genetic disorder characterized by recurring episodes of vaso-occlusive crisis (VOC) that can lead to hospitalization or sudden death. Hypoxia is an accepted trigger of sickling and degrees of nighttime hypoxia correlate with strokes and frequency of VOC. To better understand the mechanism of events leading to VOC, we simulated the occurrence of nocturnal hypoxia in SCA patients by administration of five breaths of 100% nitrogen. Tidal volume (Vt), arterial oxygen saturation, electrocardiogram (ECG), and microvascular perfusion (PU) by Laser-Doppler were continuously recorded. We had anticipated a drop in PU after each controlled episode of hypoxia. However, we observed multiple prominent drops in PU in SCA subjects (n=8) that were not as clearly evident in controls (CTL; n=9), and found no direct relationship between hypoxia and change in PU (p = NS). As deep breaths or sighs can trigger reflex peripheral vasoconstriction, we examined Vt respiratory tracings obtained simultaneously and observed that PU drops frequently followed sighs (see Figure) in SCA subjects, but rarely in CTL. A statistical algorithm was used to find all sighs and vasoconstrictive events (PU drops) during each 40-minute experimental session. PU drops were associated with sighs in 7 of 8 SCA patients and in 4 of 9 CTL subjects (P < 0.001, Poisson regression analysis). Five CTL and 1 SCA subjects had infrequent sighs and no association between sighs and PU drops. The likelihood ratio of sigh-associated PU drops was significantly higher in SCA than CTL subjects (median = 59.9 % vs. < 1 % for SCA vs. CTL, P = 0.008, rank-sum test) whereas the frequency of sighs was not significantly different between the two groups (median = 2.2 % vs. 1.3 % for SCA vs. CTL, P = 0.16, rank-sum test), indicating that SCA patients are much more likely to have sigh-associated peripheral vasoconstriction. Since the sigh-vasoconstrictor response is controlled by the autonomic nervous system (ANS), we measured heart rate variability (HRV) which is an accepted index of sympathetic/parasympathetic balance. These studies showed substantial reduction of parasympathetic modulation of HRV during hypoxia in SCA but not in CTL subjects (p < 0.01), indicating a marked abnormality of the ANS in SCA. In overview, the likelihood of coupling between spontaneous sighs and subsequent vasoconstrictive events (PU drops) is much higher in SCA patients than in CTL. Thus, we speculate that a drop in perfusion secondary to increased neural coupling between the lung and vasculature may be an initiating event in VOC. Hypoxia may secondarily promote VOC by altering ANS sensitivity and increasing the probability that a sigh will in turn lead to reflex peripheral vasoconstriction. In a background of HbS, transient decreases in perfusion may prolong red cell residence time in the microvasculature, leading to HbS polymerization, sickling and vascular occlusion. Disclosures No relevant conflicts of interest to declare.

Elevated Systolic Blood Pressure and Low Fetal Hemoglobin Are Risk Factors for Silent Cerebral Infarcts in Children with Sickle Cell Anemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 262-262
Author(s):  
Sharada A. Sarnaik ◽  
James F. Casella ◽  
Bruce A Barton ◽  
Michele Afif ◽  
Gladstone Airewele ◽  
...  
Keyword(s):  
Risk Factors ◽  
Blood Pressure ◽  
Systolic Blood Pressure ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Conflicts Of Interest ◽  
Beta Thalassemia ◽  
Cerebral Infarcts ◽  
Hemoglobin F ◽  
Elevated Systolic Blood Pressure

Abstract Abstract 262 Introduction: The most common cause of neurological injury in sickle cell anemia is silent cerebral infarcts (SCI). In the Silent Cerebral Infarct Multi-Center Clinical Trial (SIT Trial) cohort, we sought to identify risk factors associated with SCI. Patients and Methods: In this cross-sectional study, we evaluated the clinical history, baseline laboratory values and performed magnetic resonance imaging of the brain. For those children with SCI-like lesions, a pediatric neurologist examined the child and neuroradiology and neurology committees adjudicated the presence of SCI. Children between the ages of 5 and 15 years with hemoglobin SS or S-beta° thalassemia and no history of overt strokes or seizure were evaluated. Results: A total of 542 children were evaluated; 173 (31.9%) had SCI. The mean age of the children was 9.3 years, with 280 males (51.7%). In a multivariate logistic analysis, two covariates were significant: a single systolic blood pressure (SBP) obtained during a baseline well-visit, p = 0.015 and hemoglobin F (Hgb F) level obtained after three years of age, p = 0.038. Higher values of SBP and lower values of Hgb F increased the odds of SCI; Figure. Baseline values of white blood cell count, hemoglobin level, oxygen saturation, reticulocytes, pain, or ACS event rates were not associated with SCI. Conclusion: SBP and Hgb F level are two previously unidentified risk factors for SCI in children with sickle cell disease. Modulation of SBP and Hgb F levels might decrease the risk of SCI. Disclosures: No relevant conflicts of interest to declare.

A Novel Connection Between Stress Erythropoiesis and Coagulation Activation in Sickle Cell Disease.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 905-905
Author(s):  
Julia E. Brittain ◽  
David Manly ◽  
Leslie V. Parise ◽  
Nigel Mackman ◽  
Kenneth I. Ataga
Keyword(s):  
Flow Cytometry ◽  
Sickle Cell Disease ◽  
Tissue Factor ◽  
Sickle Cell ◽  
Whole Blood ◽  
Conflicts Of Interest ◽  
Cell Disease ◽  
Coagulation Activation ◽  
Monocytic Cells ◽  
Stress Erythropoiesis

Abstract Abstract 905 Introduction: Sickle cell disease (SCD) is associated with a hypercoagulable state. Multiple studies show that plasma from these patients exhibit: 1) increased thrombin generation; 2) decreased levels of natural anticoagulant proteins; and 3) a defect in the activation of fibrinolysis. The mechanism of coagulation activation in SCD is presumed to be multi-factorial, with contributions from abnormal erythrocyte phospholipid asymmetry and induction of tissue factor (TF) following hemolysis. In addition, hemolysis in SCD leads to elevated levels of erythropoietin (EPO) in patients, increased reticulocyte counts and the presence of stress (or shift) reticulocytes in circulating blood. These stress reticulocytes retain expression of the α4b1 integrin and are demonstrably adhesive to vascular factors in SCD. We have previously reported that these stress reticulocytes bind to blood monocytes in SCD patients via the α4b1 integrin, but the effect of this interaction on either cell remained unknown in SCD. Objective: With the increasing evidence that hemolysis and subsequent stress erythropoiesis associates with coagulation activation, we sought to evaluate the role of erythropoietin and the effect of stress reticulocyte adhesion to monocytes on coagulation activation in SCD patients. Methods: Coagulation activation in plasma samples was examined by evaluating TF activity on microparticles derived from patients with SCD. Stress reticulocytes were visualized and enumerated from these same patients using Wright Giemsa stained blood smears counter stained with new methylene blue to detect reticulocytes. Reticulocytes were scored as a stress reticulocytes based on the amount of punctuate reticular material, cell size, and presence of nuclear material. Stress reticulocyte induction of monocyte tissue factor expression was measured by flow cytometry after incubation of THP-1 monocytic cells with purified SS RBCs or control RBCs. To determine if induced THP-1 TF expression was due stress reticulocyte binding, THP-1 TF expression was examined in the presence or absence of known inhibitors of the monocyte/stress reticulocyte interaction. TF expression on CD14+ monocytes was examined in whole blood from SCD patients using flow cytometry. Plasma erythropoietin levels were quantified by ELISA. Results: We found that direct binding of the stress reticulocyte increased THP-1 TF expression 2.5 fold. This increase in TF expression was completely ablated by function blocking antibodies against the α4 integrin, but not by an isotype-matched control IgG. In whole blood samples, we also found increased TF expression on CD14+ monocytes with stress reticulocytes directly bound, compared to those monocytes in the same patient without stress reticulocytes bound (p = 0.002, n =3).We noted a strong correlation between stress reticulocyte count and TF activity on plasma microparticles in SCD (rspearman = 0.8656, CI = 0.5382 – 0.9660, p = 0.0006, n=11). Furthermore, we found that EPO induced α4b1 activation on the stress reticulocyte. This activation may promote both adhesion to the monocyte and an increase in TF expression. Consequently, we noted a strong trend towards an association of EPO with microparticle TF activity in SCD (rspearman = 0.5740, CI=-0.06 – 0.8780, p=0.068, n= 11) suggesting that EPO, by promoting the interaction between the stress reticulocyte and the monocyte, may contribute to TF activity in SCD. Conclusion: Taken together, we find that stress reticulocyte adhesion to monocytes and monocytic cells induces TF expression and may promote TF activity in patients. These data suggest a novel connection between stress erythropoiesis and coagulation activation in SCD. Disclosures: No relevant conflicts of interest to declare.

Sleep In Patients with Sickle Cell Anemia and Priapism. A Case-Control Study

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1644-1644
Author(s):  
Marina Roizenblatt ◽  
Frederico Pollack-Filho ◽  
Joao R Sato ◽  
Perla Vicari ◽  
Francisca Veloso ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Rem Sleep ◽  
Case Control Study ◽  
Conflicts Of Interest ◽  
Case Control ◽  
Apnea Hypopnea Index ◽  
Penile Tumescence ◽  
Control Study ◽  
Overnight Polysomnography

Abstract Abstract 1644 Background. Priapism has been described as a sickle cell anemia (SCA) manifestation that occurs frequently during sleep. Given that hypoxemia has been implicated in the pathogenesis of SCA complications, we conducted this case control study of overnight polysomnography and penile tumescence recording in SCA adults with and without priapism, with the hypothesis that SCA patients with priapism might exhibit apnea and oxyhemoglobin desaturation during sleep. Objectives. The primary endpoint was to determine the relation between increase in penile tumescence (of 20%) and respiratory sleep events (apnea, hypopnea and desaturation >3%). Further sleep event associations with penile tumescence variation and other differences between groups regarding sleep pattern were assessed as secondary endpoints. Methods. Seventeen SCA adults with priapism and 14 without history during the previous 12 month period were submitted to clinical interview and overnight polysomnography (Somnologica, Embla™), concomitantly performed with penile tumescence recording (Rigiscan Plus, Timm Medical™). Results and Discussion. There was no statistical difference between groups (t test, Chi2 or Fisher) in terms of age (mean ± s.d., 31.2 ± 14.0 vs. 27.8 ± 7.0 yrs, p=0.39), body mass index (20.9 ± 3.7 vs. 21.4 ± 2.3 kg/m2, p=0.66), use of hydroxyurea (41 vs. 43%, p=1.00), levels of ferritin (257.2 ± 291.8 vs. 429.1 ± 410.8 mg/L, p=0.27), hemoglobin (10.0 ± 1.9 vs. 9.4 ± 1.7 mg/dL, p=0.45) and hematocrit (27.4 ± 8.3 vs. 27.0 ± 5.3 %, p=0.89). No difference was also observed in sleep architecture and number of events of increase in penile tumescence, both in non rapid eye movement (non REM) and REM sleep (21.9 ± 6.8 vs. 18.2 ± 7.6, p=0.17 and 7.5 ± 3.3 vs. 8.3 ± 5.0, p=0.59, respectively). Coincidence of these events with apnea-hypopnea, desaturation and periodic leg movement (PLM) events in non REM sleep was greater in SCA patients with priapism than in those without it (16.9 ± 11.1 vs. 7.9 ± 7.1, respectively, p=0.01). PLM index was increased in both groups, particularly in SCA patients with priapism (16.1 ± 16.2 vs. 5.7 ± 5.4 /h, p= 0.03) and associated with restless leg syndrome (88.2 vs. 50.0 %, respectively, p=0.02). Desaturation index was also higher in priapism group, in comparison to non-priapism (12.7 ± 8.8 vs. 7.6 ± 3.6 /h, respectively, p=0.05), as well as apnea hypopnea index (12.2 ± 11.0 vs. 5.5 ± 4.5 /h, respectively, p=0.04). Conclusion. Compared to the group without priapism, SCA patients with priapism presented increase in apnea hypopnea index, PLM indexes during sleep and also association of increase in penile tumescence with respiratory events and PLM during non REM sleep. Our findings reinforce the literature data which highlights the link between disordered sleep and vaso-occlusive events in SCA. Moreover, the high frequency of PLM in SCA patients could be related to lower hepcidin levels recently observed in both SCA and restless legs syndrome. FAPESP CEPID 98/14303-3 and AFIP. Disclosures: No relevant conflicts of interest to declare.

Sequence-Specific Conversion of βA- to βS-Globin by Small Fragment Homologous Replacement In Hematopoietic Stem/Progenitor Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3754-3754
Author(s):  
Alireza Abdolmohammadi ◽  
Rosalie Maurisse ◽  
Babek Bedayat ◽  
David DeSemir ◽  
Damian Laber ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Dna Sequences ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Specific Gene ◽  
Small Fragment ◽  
Wild Type ◽  
Hematopoietic Stem

Abstract Abstract 3754 Introduction: An ultimate goal of gene therapy is the development of effective strategies to correct mutant genomic sequences in pathologic cells. To that end, studies have been undertaken to evaluate the therapeutic potential of an oligo/polynucleotide-based sequence-specific gene modification strategy, small fragment homologous replacement (SFHR) in the correction of the mutation giving rise to sickle cell anemia. Small DNA fragments (SDFs) comprising the sickle cell anemia mutation (an A>T transversion in codon 6) and flanking DNA sequences in the human b-globin gene were introduced into Hematopoietic Stem/Progenitor Cells (HSPCs). The studies presented indicated modification at the level of DNA, RNA, and protein when cells were differentiated into erythrocytes. Methods: In this study, SFHR was used to convert A>T in codon 6 of the b-globin gene in CD34+/CD38-/Lin- HSPCs isolated from full term umbilical cord blood as a proof of principle. HSPCs were transfected with a defined number of a 559-bp SDF using the Amaxa electroporation (nucleofection) system. After growing the transfected cells in stem cell media containing EPO for different time intervals up to 32 days, RNA was extracted and DNase I-treated before further analysis. Erythrocytes were also analyzed using antibodies that differentiate between wild-type hemoglobin A (HBA) and sickle cell hemoglobin S (HBS). Results: RFLP analysis of a 430-bp PCR product generated from mRNA-derived cDNA with the DdeI enzyme indicated conversion of bA- to bS-globin. Sequencing of the 430-bp amplicon showed the A > T conversion. Analysis of the transfected wild-type HSPC-derived erythrocytes with HBA and HBS specific antibodies demonstrated the presence of a subpopulation of cells expressing HBS. These data are consistent with previous studies showing both conversion of bS- to bA-globin in SC1 cells and bA- to bS-globin in HSPCs after electroporation and microinjection of SDF, respectively. Conclusion: These studies represent a critical next step in developing SFHR as a therapy for sickle cell disease, in that they demonstrate long-term SFHR-mediated modification of b-globin following mass transfection by electroporation of HSPCs. Disclosures: No relevant conflicts of interest to declare.

Serum Transferrin: An Independent Predictor of Mortality in Sickle Cell Anemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2126-2126
Author(s):  
Zahra Pakbaz ◽  
Mariana E Hildesheim ◽  
Shoaib Alam ◽  
Darlene Allen ◽  
Caterina Minniti ◽  
...  
Keyword(s):  
Endothelial Dysfunction ◽  
Sickle Cell ◽  
Serum Ferritin ◽  
Sickle Cell Anemia ◽  
Conflicts Of Interest ◽  
Independent Predictor ◽  
Stable Condition ◽  
Acute Chest Syndrome ◽  
Serum Transferrin ◽  
Significant Difference

Abstract Abstract 2126 Introduction: Serum ferritin (SF), the most convenient marker of iron burden in sickle cell anemia (SCA), is potentially confounded by effects of inflammation in SCA. Serum transferrin (STF) has been described as one of the independent predictors of elevated tricuspid regurgitant velocity (TRV) in SCA. Therefore in this report we investigate the potential predictive role of STF in morbidity and mortality of individuals with SCA. Methods: Patients with sickle cell disease documented by high-pressure liquid chromatography were eligible for the study. Four hundred and sixty SCA patients were recruited in this study from the community through multimedia advertisements, community outreach, and regional clinics. All evaluated patients were screened by history taking, physical examination, laboratory studies, and transthoracic echocardiography. All patients provided written informed consent. The advertisements and protocol were approved by the institutional review boards of the National Heart, Lung, and Blood Institute and Howard University. Only outpatients in stable condition were included; patients who had had a vaso-occlusive crisis within the previous two weeks or an episode of acute chest syndrome within the previous four weeks were excluded. Results: Two hundred and sixty two participants with HbSS were included in the data analysis. Forty-seven percent were male. Median age was 32 years old. Median TRV in this cohort was 2.4 m/s. Forty nine percent of participants had TRV≥2.5 m/s and 19% had TRV≥3 m/s. Patients with lower STF (<164 mg/dl) were older (p=0.01), had more blood transfusions in the past (p<0.0001) and did not have more complications of sickle disease (priapism, acute chest syndrome, leg ulcers or emergency room visits, all p>0.05), but all-cause death rate was higher (22% vs. 6%, p=0.0001). Hemoglobin, CRP, alkaline phosphatase, uric acid, placenta growth factor (<0.001), SF and iron saturation were found to be higher and kidney function was worse. Patients with lower STF level were also more likely to have TRV≥3.0 m/s (32% vs. 13%, p<0. 001) but there was no significant difference in BNP and ejection fraction. Patients with lower STF had endothelial dysfunction, as indicated by a blunted forearm blood flow (FBF) response to infusion of acetylcholine into the brachial artery (p<0.01). Among age, gender, BNP, TRV,GFR,WBC, STF, systolic blood pressure and fetal hemoglobin, the Cox proportional analysis of mortality found TRV, GFR and STF the independent significant predictors of mortality in this cohort. Kaplan-Meier survival curve showed that patients with transferrin <164 mg/dl had significantly lower survival (p<0.001). Conclusion: In this cohort of adults with sickle cells anemia, STF is found to be an independent predictor of endothelial dysfunction, high TRV and mortality. It may be a more sensitive predictor than serum ferritin. We propose that iron overload may induce a state of endothelial dysfunction that is a risk factor for clinical vasculopathy and death. Disclosures: No relevant conflicts of interest to declare.

Identification of Genetic Modifiers Associated with Risk of Stroke in Children with Sickle Cell Anemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3228-3228
Author(s):  
Jonathan Michael Flanagan ◽  
Heidi Linder ◽  
Vivien Sheehan ◽  
Thad A Howard ◽  
Banu Aygun ◽  
...  
Keyword(s):  
Cerebrovascular Disease ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Conflicts Of Interest ◽  
Association Studies ◽  
Snp Markers ◽  
Genome Wide Association Studies ◽  
Genetic Modifiers ◽  
Sibling Pairs ◽  
Synonymous Mutations

Abstract Abstract 3228 Introduction: Stroke is one of the most catastrophic acute complications of sickle cell anemia (SCA), occurring in 11% of patients before 20 years of age. A further 20 to 30% of children with SCA will develop less clinically overt cerebrovascular disease events such as transient ischemic attacks (TIA) and silent infarcts. There is a definite need for biomarkers that could determine the cause of these irreversible cerebrovascular events and which might predict children at greatest risk. Previous studies of sibling pairs have shown that there is a genetic component to cerebrovascular disease development but few genetic modifiers have been validated as having a substantial effect on risk of stroke. The aim of this study was to perform an unbiased whole genome search for genetic modifiers of stroke risk in SCA. Methods: Pediatric patients with SCA and documented primary stroke (n=177) were compared to a pediatric control non-stroke group with SCA (n=335). All control patients were over 5 years old and without previous clinical stroke prior to beginning any clinical treatment. Genome wide association studies (GWAS) were performed using genotype data obtained from Affymetrix SNP6.0 arrays. A pooled DNA approach was used to perform whole exome sequencing (WES) by Illumina next generation sequencing of pooled control (n=104) and pooled stroke (n=120) groups. Results: From the Affymetrix SNP6.0 GWAS data, 139 single nucleotide polymorphisms (SNP) were identified with stroke association. From the WES, 294 non-synonymous mutations were found to be significantly associated with stroke. In combination, 11 mutations identified by WES were located within 250kb of a SNP identified by GWAS (Table 1). These 11 mutations represent key areas of the genome that are targets for further in depth study. To next validate the genetic variants identified by WES with association with risk of stroke, 21 candidate mutations were genotyped in an independent cohort of control (n=231) and stroke (n=57) patients with SCA. One mutation in GOLGB1 (Y1212C) was corroborated as having significant association with lower risk of stroke (p=0.02). Conclusion: This mutation in GOLGB1 is predicted to effect the golgi associated function of the encoded protein and future studies will focus on how this functional mutation may protect against development of cerebrovascular disease in the context of SCA. For all variants with significant association with stroke, the chromosomal position of each variant identified by WES (n=300, p<0.001) was compared to the location of all SNP markers (n=139, p<0.0001). We identified 11 variants by WES where there was at least one SNP marker within 250kb. These variants all represent excellent regions of the genome for future study. The four variants highlighted with a asterisk (*) are variants predicted by PolyPhen2 or SIFT to be deleterious. Disclosures: No relevant conflicts of interest to declare.

Monocyte Shift to a Non-Classical CD14dim/CD16+ Phenotype Correlates with Fetal Hemoglobin Levels in Sickle Cell Anemia Patients Treated with Hydroxyurea

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 817-817 ◽  
Author(s):  
Kleber Yotsumoto Fertrin ◽  
Dulcinéia Martins Albuquerque ◽  
Carolina Lanaro ◽  
Carla Fernanda Franco-Penteado ◽  
Flavia Rubia Pallis ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Cell Activation ◽  
Conflicts Of Interest ◽  
Fetal Hemoglobin ◽  
Pleiotropic Effect ◽  
Monocyte Subset ◽  
Classical Monocytes ◽  
Intermediate Monocytes

Abstract Abstract 817 Vaso-occlusion in sickle cell anemia (SCA) involves inflammation and cell activation; fetal hemoglobin (HbF) elevation by hydroxyurea (HU) remains the mainstay of SCA treatment. Monocytes are activated in SCA, and their contribution to the chronic inflammatory state includes the production of cytokines and reactive oxygen species (ROS). Monocytes are a heterogeneous group of leukocytes subdivided into distinct subsets: classical monocytes comprise over 80% of circulating monocytes, are highly positive for CD14 (CD14bright) and typically CD16-negative, while CD16-positive monocytes have been further subdivided into intermediate CD14bright/CD16+, and non-classical CD14dim/CD16+ monocytes. Intermediate monocytes are recognized as the main monocytic producers of ROS and are increased in inflammatory conditions such as atherosclerosis and sepsis. The less characterized non-classical subset is believed to have a patrolling behavior in blood vessels, does not produce ROS and constitutively produces IL-1 receptor antagonist (IL1-RA). Another relevant subgroup of monocytes expresses angiopoietin-2 receptor TIE2, and the role of TIE2-expressing monocytes (TEMs) has been investigated in angiogenesis in neoplastic diseases. TEMs usually correspond to intermediate monocytes, but their importance in inflammation is still unclear. We hypothesized that monocyte subsets in SCA patients would differ from controls, and that treatment with HU might also influence monocyte phenotypes, thus shedding light on the possible role of these subsets in an inflammatory condition not previously studied. EDTA-anticoagulated peripheral blood samples were collected upon written informed consent from 21 healthy controls (CON, ages 21–63 years) and 34 SCA patients (18 on HU, ages 16–58 years) in steady state, with no transfusion or acute sickling episode in the previous three months. Monocytes were immunophenotyped by flow cytometry on a multicolor FACSCalibur cytometer. Medical history of SCA-associated complications, HbF levels and dosage of HU in mg/kg/day were obtained from medical charts. Statistical analysis was performed on GraphPad Prism 5.0 software. As expected, we found that relative percentage and absolute count of CD16-positive monocytes were higher in SCA patients than in controls. Surprisingly, a significantly higher percentage of non-classical CD14dim/CD16+ monocytes, rather than intermediate cells, was found in SCA patients on HU (SCA-HU) treatment (mean±SEM: CON 2.06±0.43%, SCA 2.91±0.50%, SCA-HU 6.42±0.80%, P<0.0001). TEMs were also increased in SCA patients compared to controls (CON 2.64±0.72%, SCA 20.48±5.40%, SCA-HU 32.97±5.92%, P<0.0001), but HU treatment did not significantly influence TEM counts. Mean TIE2 expression did not vary among the groups, and there was no correlation between TEMs and presence of SCA complications pathophysiologically associated with disturbed angiogenesis, such as pulmonary hypertension, osteonecrosis, leg ulcers and retinopathy. Higher percentages of non-classical monocytes in HU-treated patients were initially interpreted as a possible toxic effect of HU on monocytopoiesis, but the lack of correlation of monocytes subsets with the degree of relative monocytopenia made this hypothesis unlikely. Moreover, we found a significant positive correlation between percentages of non-classical monocytes and HbF levels (rS=0.4763, P=0.0068, see figure). This suggests that successful HU treatment with higher HbF could correlate with the expansion of this particular monocyte subset. During the study period, only one patient was available for comparison before and after HU, but the increase in HbF from 4.2% to 11.6% and in non-classical monocytes from 1.82% to 9.48%, in this case, corroborates that HU therapy may explain this phenotype shift in monocytes. Whether non-classical monocytes expansion represents yet another pleiotropic effect of HU, if these cells are less likely to take part in the vaso-occlusive process and have an antiinflammatory role or, furthermore, if a bone marrow counterpart of this monocyte subset could be involved in increasing HbF production, remains to be investigated. The correlation of the expansion of non-classical monocytes with HbF levels could prove to be an interesting biomarker of response to HU, and future studies may address its clinical usefulness. Disclosures: No relevant conflicts of interest to declare.

Hemoglobinuria Is a Risk Factor For Kidney Disease Progression In Sickle Cell Anemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 996-996
Author(s):  
Santosh L. Saraf ◽  
Xu Zhang ◽  
Tamir Kanias ◽  
James P. Lash ◽  
Robert E. Molokie ◽  
...  
Keyword(s):  
Steady State ◽  
Kidney Disease ◽  
Kidney Function ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Conflicts Of Interest ◽  
Red Blood Cell Transfusion ◽  
Free Hemoglobin ◽  
Arterial Blood

Abstract Chronic kidney disease (CKD) is a frequent complication of sickle cell anemia (SCA) and is a predictor of early mortality. To determine the predictors of deteriorating kidney function in SCA, we followed 164 patients treated at the University of Illinois at Chicago for a median of 32 months (range 3-88 months). Steady-state estimated glomerular filtration (eGFR), albuminuria, and hemoglobinuria assessments were obtained at baseline and during the follow-up period. Steady-state was defined as greater than four weeks from a vaso-occlusive pain episode or a red blood cell transfusion. Hemoglobinuria was defined as positive for blood on dipstick and < 2 red blood cells on microscopy. Fifty-six (34%) of the patients had hemoglobinuria at baseline. We confirmed in a subset of 43 patients that dipstick positive hemoglobinuria (n=17) was associated with higher urine cell-free hemoglobin concentrations determined by ELISA than dipstick negative urine (n=26) (23.1 vs. 11.5 ng/mL, p<0.0001) (Figure 1). Age and mean arterial blood pressures were similar in patients with hemoglobinuria at baseline compared to those without but markers of hemolysis were higher (LDH, indirect bilirubin, AST, and reticulocyte percentage; p<0.0001). Sixty-one percent (95%CI: 48-73%) of patients with hemoglobinuria at baseline had hemoglobinuria at most recent follow up compared to 9% (95%CI: 5-18%) of patients without hemoglobinuria at baseline (p<0.0001). The proportion of patients with CKD progression defined by a 50% reduction in eGFR calculated by the CKD-EPI formula or requirement for hemodialysis or kidney transplant was higher in patients with baseline hemoglobinuria (13%, 7/56) versus without hemoglobinuria (1%, 1/108) (HR 14, 95%CI: 2-113; logrank p=0.001) (Figure 2). Progression of albuminuria category from normoalbuminuria (albuminuria < 30mg/g creatinine) to either microalbuminuria (albuminuria = 30-300 mg/g creatinine) or macroalbuminuria (albuminuria > 300mg/g creatinine) or microalbuminuria to macroalbuminuria was also higher in patients with baseline hemoglobinuria (42%, 11/26) versus without hemoglobinuria (13%, 9/67) (HR 3.1, 95%CI: 1.3-7.7; logrank p=0.004) (Figure 3). In conclusion, hemoglobinuria determined by urinalysis at steady-state is a valid assessment of increased urine cell-free hemoglobin concentration and is fairly consistent on repeat testing at steady-state visits. The presence of hemoglobinuria is significantly associated with a greater risk for progression of CKD and albuminuria. Our findings are consistent with the possibility that cell-free hemoglobin contributes to the progression of kidney disease in SCA. Further research including measures to decrease cell-free hemoglobin exposure to preserve kidney function are warranted.Figure 1Figure 1. Figure 2Figure 2. Figure 3Figure 3. Disclosures: No relevant conflicts of interest to declare.

Increased Platelet-Derived CD40L May Modulate Endothelial Cell ICAM-1 Expression and Interact With Leukocytes In Sickle Cell Anemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 974-974
Author(s):  
Vanessa Tonin Garrido ◽  
Renata Proença-Ferreira ◽  
Venina M. Dominical ◽  
Marcos André Cavalcanti Bezerra ◽  
Aderson S. Araujo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Steady State ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Repeated Measures ◽  
Conflicts Of Interest ◽  
Endothelial Activation ◽  
Surface Expression ◽  
Platelet Surface

Abstract Background Vaso-occlusive events are a major cause of morbidity in sickle cell anemia (SCA) and attributable to the abnormal adhesion of red cells and leukocytes to the endothelium. Platelets may contribute to the chronic inflammation and endothelial activation that initiates the vaso-occlusive process. We hypothesized that platelet-associated CD40 ligand (CD40L) may contribute to platelet-mediated inflammatory responses in SCA. Aims This study evaluated the platelet (PLT) release of CD40L, the expression of its receptor (CD40) on platelets, neutrophils, lymphocytes and monocytes of control individuals (CON) and SCA patients, and also the ability of platelet-derived CD40L to activate endothelial cells. Methods IL-8, soluble ICAM-1, VCAM-1 and CD40L were determined in PLT-free plasma or the supernatant of stimulated (ADP or Collagen) and unstimulated PLTs (2•10⁸/mL in Kreb’s buffer), from CON individuals and steady-state SCA patients, by ELISA. Flow cytometry was used to analyze CD40 expression on platelets, neutrophils, lymphocytes and monocytes from the peripheral blood of the study’s subjects. Human umbilical vein endothelial cells (HUVECs) were cultured (1x106cells/well; 37°C, 5% CO2) together with PLTs (3x108PLTs/well) from CON individuals or steady-state SCA patients for 24h, 37°C, 5%CO2, in the presence, or not, of blocking antibodies against CD40L. After incubation, PLTs were removed and HUVECs analyzed by flow cytometry for CD54 (ICAM-1) surface expression. Results SCA individuals presented elevated levels of plasma CD40L (724.4± 55.7 pg/ml; n=90) compared to CON (241.5±34.6 pg/ml; n=41; P<0.0001) and these levels correlated with PLT counts (rs=0.255; P=0.015). No correlation was found between plasma CD40L and plasma IL-8, ICAM-1 or VCAM-1. PLT release of CD40L (90 min, 37°C, 5%CO2) was evaluated; PLTs of SCA patients released higher quantities of CD40L (8347±1464 pg/108 PLTs; n=10) than PLTs of CON individuals (3652±568 pg/108 PLTs; n=5; P=0.019). CD40L release from SCA PLTs was augmented by incubation with collagen (P<0.001), but not ADP. Expression of the CD40 receptor on the platelet surface was elevated in the SCA group (52.4±2.7% positive cells; n=23), compared to the CON group (36.8±3.7% positive cells; n=9; P=0.005). The surface expression of CD40 was also elevated on neutrophils (SCA, 10.4±1.5% positive cells, n=14; CON, 5.5±1.1% positive cells, n=13; P=0.03), lymphocytes (SCA, 8.3±0.8% positive cells, n=16; CON, 3.6±0.4% positive cells, n=14; P<0.001) and monocytes (SCA 69.6±5.9% positive cells, n=16; CON, 49.9±5.8% positive cells, n=14; P=0.03) of SCA patients, compared to controls. ICAM-1 expression on the surface of HUVECs (Basal expression 32.8±1.8%, n=11) was significantly increased following incubation with SCA PLTs (54.0±4.8%, n=11, p<0.0001) and slightly augmented after incubation with CON PLTs (40.8±3.1%, n=11, p<0.05; Repeated measures ANOVA). Interestingly, when HUVECs and SCA PLTs were incubated with a blocking antibody against CD40L, the increase in ICAM-1 expression was significantly reversed on HUVECs (HUVECs, 28.1±0.2%, n=6; HUVECs+SCA PLTs, 42.0±3.3%, n=6; HUVECs+SCA PLTs+anti-CD40L 28.9±1.5%, n=6; P<0.01). Conclusions Plasma levels and platelet release of CD40L were found to be significantly elevated in SCA, in association with increased expressions of the CD40 receptor on SCA PLTs, neutrophils, lymphocytes and monocytes, possibly indicating a CD40L-mediated crosstalk between platelets and leukocytes in SCA. Platelets from SCA patients can induce adhesion molecule expression on the surface of endothelial cells in vitro, and this up-regulation may be modulated by platelet-derived CD40L. Results suggest that the CD40/CD40L pathway may be altered in SCA and that platelets may participate in this up-regulation. Given the potent inflammatory effect of this cytokine, a role for platelets and this cytokine in endothelial activation, inflammation and consequent vaso-occlusion, is likely. Disclosures: No relevant conflicts of interest to declare.

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