ALBERT

Description: Background: Sickle cell disease (SCD) is a chronic illness associated with varying degrees of decrease in exercise capacity. Affected children are as much as 50% less physically active than controls. The reasons for the reluctance of SCD children to participate in sports and/or intense physical activities are complex, not entirely understood and not well studied in this young population with chronic anemia. The six minute walk test (6MWT) is an exercise test, validated as screening tool for assessing cardiopulmonary functional status in several pediatric cohorts of healthy children and in children with chronic illnesses including SCD. This test is a submaximal exercise test and reflects the patient’s capacity to undertake daily activities. We have previously reported that children with SCD experience a decrease in oxy-hemoglobin oxygen saturation as determined by pulse oximetry after the test. Material and methods: We performed a prospective, multicenter study of cardiopulmonary function in 409 children with SCD (age 5-20 years) and 70 age and race matched controls as part of the PUSH study. Each subject was at steady state, at least three weeks from an acute exacerbation and/or a blood transfusion. Subjects and controls underwent echocardiogram, hematologic testing, and 6MWT according to standard protocols. 6MW distance was compared between SCD cases and controls by the student t-test. A linear regression model was developed to assess the independent predictors of 6MW in patients. Results: SCD patients had lower 6MW distance (p

Description: Abstract 1511 Poster Board I-534 Background What determines the degree of hemolysis and of anemia in patients with hemoglobin SS is not fully known. The rate of hemolysis and severity of anemia are ameliorated by the presence of alpha thalassemia and by higher hemoglobin F percentage. Mild G6PD deficiency in the form of G6PD-202/-376 may be associated with episodic hemolysis in individuals of African descent, but past studies indicated little influence of G6PD-202/-376 on the degree of hemolysis and anemia in sickle cell disease patients (1,2). In this study we examined the roles of single and double α-globin deletions and G6PD-202/-376 on the degree of hemolysis and the hemoglobin concentration in hemoglobin SS patients. Methods Two hundred sixty two children and adolescents with hemoglobin SS were recruited at three tertiary medical centers and studied at steady state. Principal component analysis was used to develop a hemolytic component from concentrations of lactate dehydrogenase, aspartate aminotransferase and bilirubin. PCR was used to determine the presence of α-thalassemia and G6PD-202/-376. Multivariate models were employed to determine the independent effects of these genotypes on hemoglobin concentration and degree of hemolysis. Results Single a-globin deletion was associated with an estimated 0.4 g/dL increase in steady-state hemoglobin concentration and double α-globin gene deletion with a 0.8 g/dL increase (P = 0.005 for trend) due to, progressively lower degrees of hemolysis (P = 0.004). G6PD-202/-376 was associated with an estimated 0.7 g/dL decrease in the hemoglobin concentration (P = 0.003) (Figure 1a), but this observation could not be explained by increased hemolysis. Rather, the reticulocyte count was an estimated 22% lower with G6PD-202/-376 (P = 0.032) (Figure 1b). Discussion G6PD -202/-376 may be associated with lower hemoglobin concentration in sickle cell anemia and the mechanism is probably impaired erythropoiesis rather than hemolysis. A recent study (3) indicates that G6PD is needed for definitive erythropoiesis as well as for normal survival of red blood cells in the periphery. Our present findings raise the possibility that, in the setting of the markedly increased erythropoiesis of sickle cell anemia, G6PD-202/-376 may result in impairment in erythropoiesis that is discernible in the peripheral blood hemoglobin concentration. Disclosures No relevant conflicts of interest to declare.

Description: BABY HUG (NCT00006400) is an NHLBI-NICHD sponsored Phase III multi-center, randomized, double blind placebo controlled study of daily oral hydroxyurea in infants 9–17 months of age with sickle cell anemia. Subject retention and adherence are essential to maintain the power and validity of the trial. Initially, we hypothesized that families with higher socioeconomic status (SES) and advanced education would be better able to comply with the complex study procedures. We evaluated SES and other demographic factors which may influence adherence in the ongoing study. Both study drug adherence and visit adherence were examined. Study visits occurred every two weeks until a stable dose was reached and then every four weeks unless toxicity was encountered. Subjects remained on study for two years. Study drug adherence was monitored by measuring liquid in returned bottles at each visit. Bottles were included in the analysis if the subject was dispensed a bottle at a visit, returned it at the next scheduled visit with a non-zero volume, and did not have a drug stop order between the visits. From these bottles, each subject’s median drug adherence was calculated. Drug adherence (%) was defined as the amount consumed divided by the amount which should have been consumed. Visit adherence (%) was based on expected routine study visits. Subjects were withdrawn from the study upon family request or if they continually missed visits. We defined ‘good adherence’ as taking ≥80% study drug and attending ≥90% study visits. Adherence data was available on 186 of the 193 randomized infants. The median drug adherence was 101.7%, with 88.9% of subjects having drug adherence of ≥80%, and three infants having drug adherence 〉150%. The mean visit adherence was 97.3% ± 8.4 SD, with 82.3 % of subjects having no missed visits. Thirteen infants had a visit adherence of

Description: Abstract 3216 Fetal hemoglobin (HbF) is the major genetic modifier of clinical course of sickle cell anemia (homozygosity for HBB glu6val). HbF level is also an important predictor of mortality. If it were possible to know at birth the HbF level likely to be present after stabilization of this measurement at about age 5 years, then an improved prognosis might be given and HbF-inducing treatments better informed. Levels of HbF in adults are highly heritable and the production of HbF is genetically regulated by several quantitative trait loci and by genetic elements linked to the HBB gene cluster. One of the most popular approaches to genetic risk prediction uses a summary of the risk alleles in the form of a genetic risk score (GRS) that is used as a covariate of the genetic prediction model. We present the development of a GRS for HbF in 841 patients from the Cooperative Study of Sickle Cell Disease (CSSCD) cohort patients and assessed its ability to predict HbF values in three independent cohorts that included PUSH (N=77), Walk-PHaSST (N=181), and C-Data from the Comprehensive Sickle Cell Centers program (N= 127). We used the results of a genome-wide association study (GWAS) of HbF in sickle cell anemia, in which patients were genotyped using the 610K Illumina array, and association of each of the ∼550K SNPs with HbF was tested using a linear regression model with gender adjusted additive genetic effects. To build the GRS, we sorted SNPs by increasing p-value, starting from the most significant SNP associated with HbF (rs766432, p-value=2.61×10−21), and pruned the list by removing SNPs in high LD (r2 〉 0.8). We then used this list of SNPs to generate a sequence of nested GRS. We started with the GRS that included only the most significant SNP and generated the second GRS by adding the second SNP from the list of SNPs. The third GRS was generated by adding the 3rd SNP from the list of SNPs to the second GRS, and so on. We repeated this analysis including up to 10,000 SNPs (p-value〈 .02185) and hence generated 10,000 GRS, for each of the subjects in the CSSCD. Each of these GRS was included as covariate in a linear regression model and the regression coefficients of the resultant 10,000 linear regression models were estimated using Least Squares methods in the CSSCD data. The predictive value of these GRS models was then evaluated in three independent cohorts. In this evaluation, we computed the 10,000 GRS for each subjects in each data sets, and then used the 10,000 regression models estimated in the CSSCD data set to compute the expected HbF value of patients, given their GRS. We then assessed the predictive accuracy by computing the correlation between the observed and predicted values of HbF. To produce more stable predictions, we also created ensembles of predictive models. An ensemble of the first 14 GRS models including 14 SNPs had the best predictive value in all 3 data sets and explains 23.4% of the variability in HbF; the correlation between the predicted HbF and observed HbF was 0.44, 0.28 and 0.39 in the three different cohorts. Of these 14 SNPs, 6 were located in BCL11A; other SNPs were located in the olfactory receptor region and the in chromosome 11p15 and the site of the HBB gene cluster and were found previously to be associated with HbF. We next compared these results to predictive models in which we included gender, coincident alpha thalassemia, and HBB haplotypes for prediction. The model including gender and alpha thalassemia explained only 2.6% of the variability of HbF in the discovery cohort and the model including HBB haplotypes explained 2.35% of the variability of HbF in the discovery cohort and neither model showed a significant correlation between the predicted and observed HbF in the three other cohorts. In addition, combining the non-genetic information with the GRS did not help to explain more of the variability in HbF. With as few as 14 SNPs we can explain more of the variability in HbF and do a better job of prediction in comparison to using other non-genetic risk factors or genome-wide significant SNPs; however, we still cannot explain all of the variability in HbF that is due to heritability. These results suggest that knowing the genotype of a few SNPs can help to predict HbF that after they have stabilized. Prediction of HbF at an early age has the potential to help foretell some features of the severity of the clinical course of the disease and aid to optimize the clinical management of patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 903 Abstract Background: Deficiency of NADH-cytochrome b5 reductase (cytb5r, EC 1.6.2.2) is responsible for congenital methemoglobinemia. This enzyme exists in soluble and membrane-bound forms. The soluble erythrocytic cytb5r isoenzyme is involved in cytochrome b5 reduction and in erythrocyte methemoglobin reduction; the membrane-bound microsomal enzyme participates in a fatty acid desaturation complex and in drug metabolism. The cytb5r isoforms are the product of a single gene locus, DIA1 (or CYB5R3), on chromosome 22. More then 40 mutations which cause methemoglobinemia have been reported to date; the majority are missense mutations and are associated with mild type I methemoglobinemia. The CYBR5 T116S mutation is the most common genetic polymorphism among African Americans known (gene frequency as high as 20%) and it has not yet been detected in other ethnic and racial groups. This polymorphism is not associated with methemoglobinemia and its functional significance is not yet known. We studied the relationship of CYBR5 T116S with the degree of hemolysis and the tricuspid regurgitation velocity (which correlates with systolic pulmonary artery pressure) in patients with sickle cell disease. Methods: Two hundred sixty one children and adolescents with hemoglobin SS were recruited at three tertiary medical centers and studied at steady state. Patients with other sickle genotypes were excluded from this analysis of CYBR5 T116S. Principal component analysis was used to develop a hemolytic component from reticulocyte count and concentrations of lactate dehydrogenase, aspartate aminotransferase and bilirubin. PCR was used to determine the presence of the CYBR5 T116S mutation. Multivariate models were employed to determine the independent effects of this genotype on degree of hemolysis and tricuspid regurgitation velocity. Results: Ninety-eight of the patients (38%) were CYBR5 T116S heterozygotes and 26 (10%) were homozygotes, consistent with Hardy-Weinberg equilibrium. Both heterozygosity (beta = -0.4) and homozygosity (beta = -0.5) were associated with reduction in the hemolytic component (N = 261; P for trend = 0.002) (Figure 1). This relationship persisted after adjusting for α-thalassemia, hemoglobin F percent and hydroxyurea treatment in a subset of 113 patients with all of this information available (P for trend = 0.037) and it also persisted in a subset of 87 patients with no α-globin gene deletion who were not being treated with hydroxyurea (P for trend = 0.029). In none of these analyses did G6PD-202/-376 have an effect on hemolysis. Both heterozygosity (beta = -0.04) and homozygosity (beta = -0.14) for the CYBR5 T116S mutation were also associated with lower tricuspid regurgitation velocity (P for trend = 0.024). Conclusions: CYBR5 T116S is a common polymorphism among patients with sickle cell disease that appears to be associated with less hemolysis and lower tricuspid regurgitation velocity. We speculate that this polymorphism may be related to a previously reported subpopulation of African Americans with increased cytochrome b5 reductase activity, and that increased anti-oxidant activity may explain the polymorphism's hemolysis-reducing effect. Functional studies to investigate this possibility are planned. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2571 Poster Board II-548 The role of iron in the pathophysiology of sickle cell disease (SCD) is complex and not fully understood. Iron overload is associated with disease severity primarily because multiple transfusions are linked to a severe SCD clinical course. Additionally, hemolysis, also associated with disease severity, increases iron absorption. Iron deficiency decreases red cell MCHC, which lowers Hb S polymerization and thus may improve the clinical manifestations of SCD. Such a hypothesis is supported by our recent observation of a homozygous SCD adult with iron deficiency anemia and a very low hemolytic rate that increased dramatically with iron supplementation. This experience and similar case reports from the literature led us to examine the relationship of ferritin levels with hemolysis and other laboratory and clinical parameters in a group of non-iron overloaded children with sickle cell disease. All subjects in this analysis were enrolled in a prospective study of the prevalence and significance of pulmonary hypertension in children with SCD (PUSH). Because of the known association of high serum ferritin with multiple transfusions and with a severe clinical course in this and other SCD populations, we excluded children who had ferritin concentrations of 242 ng/ml or higher. This cut-off value is 3 SDs above the geometric mean of the ferritin concentrations in a group of 42 age, sex, and ethnicity matched control children without SCD. Hence the group of sickle cell children with ferritin levels of 〈 242 ng/ml should include only those with iron deficiency or with normal iron stores. The table shows correlations between serum ferritin (natural log) and age, hematologic, iron status, and hemolytic parameters, including a previously described hemolytic component derived by principal component analysis from reticulocyte count, LDH, AST, and bilirubin. In this group of non-iron overloaded SCD children and adolescents (median age 12 y, range 3–20 y), lower serum ferritin was related to higher serum transferrin and to lower serum iron and MCV, documenting that serum ferritin was reflective of iron status. Hemolytic parameters such as reticulocyte count and the hemolytic component were significantly lower with lower ferritin levels. In multivariable analysis these relationships remained statistically significant (P for MCV and ferritin: 0.003, P for hemolytic component and ferritin: 0.044) even after correcting for alpha-thalassemia, which is known to also lower MCV and hemolysis, and for markers of inflammation (WBC) and liver disease (ALT), which could increase the ferritin level regardless of iron stores. Ferritin was significantly lower in older subjects, probably as a result of growth-related red cell mass expansion in the presence of marginal iron stores. Our results thus suggest that low iron stores are independently associated with decreased hemolysis. Low hemolysis is likely to be beneficial in SCD by reducing hemolysis-related vasculopathy, which in adult SCD patients predicts an increased risk of pulmonary hypertension, leg ulcers, priapism, and death. Whether iron status per se plays a role in the pathogenesis of SCD vasculopathy is not known. In non-SCD adults, decreasing iron stores by frequent blood donation has beneficial effects on endothelial function and cardiovascular disease even within the normal range for iron stores. Hence, lowering iron stores could benefit SCD subjects by an additional, hemolysis-independent mechanism. Therapeutic iron depletion is not an option for children because of their need for adequate iron stores for optimal physical and neuro-psychological development. However, carefully controlled studies should be considered to reduce iron stores and so decrease the hemolytic rate in adults with SCD. It may be possible to achieve levels of iron reduction that lower hemolysis but do not worsen the anemia: in our study subjects, low iron stores were not associated with increased anemia and the red cell counts were actually higher with lower ferritin levels. Disclosures: Gordeuk: TRF Pharma: Research Funding; Merck: Research Funding; Biomarin pharmaceutical company: Research Funding; Novartis: Speakers Bureau.

Description: Background: Sickle cell disease shares common complications such as vasculopathy and organ dysfunction involving the heart, the lungs, the liver and the kidneys with other hemolytic conditions. We hypothesized that a hemolytic vasculopathy may underlie some of these complications. Distinguishing whether a complication is due to hemolysis or to the degree of anemia has been a challenge. Methods: A prospective, multicenter study of 310 children and adolescents with sickle cell disease in steady state was conducted. The associations of measures of hemolysis and of hemoglobin concentration with disease complications were assessed. Principle component analysis was used to develop a hemolytic index from the measurements of reticulocyte count, lactate dehydrogenase, aspartate aminotransfersae and total bilirubin. In order to determine the independent associations of hemolysis with the clinical manifestations of sickle cell disease, we computed correlation coefficient of the hemolytic index with these manifestations that controlled for hemoglobin concentration. P values were adjusted for multiple comparisons. Results: Hemolysis and hemoglobin had no significant correlation with number of the severe pain episodes, acute chest syndrome and priapism. The hemolytic index correlated with history of stroke (r= 0.19, p=0.026), white blood cell count (r=0.22, p

Description: Background:The pathogenesis of pulmonary hypertension in sickle cell disease is not fully defined. We have found independent associations of hemolysis and hemoglobin oxygen desaturation with elevated tricuspid regurgitant jet velocity in a prospective multicenter study of 310 children and adolescents with sickle cell disease. The present report includes a subset of these patients in whom we investigated the association with jet velocity of an array of cytokines and biomediators that have previously been associated with vasculopathy and primary or secondary hypertension. Methods:Jet velocity was prospectively determined by Doppler echocardiograph in 237 children and adolescents with sickle cell disease at steady state. Elevated jet velocity was defined as ≥2.6 m/sec based on the mean + 2 SD in control subjects matched by age, sex and ethnicity to every sixth patient. Plasma concentrations of interleukins-6, 8 and10, interferon-γ, tumor necrosis factor-α, vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), basic fibroblast growth factor (bFGF), platelet-derived growth factor-BB (PDGF-BB) and Regulated upon Activation Normal T-cell Expressed and Secreted (RANTES) were determined by a multiplex cytokine kit and the Bio-Plex suspension array system (Bio-Rad, Hercules, CA). Plasma concentrations of endothelin-1 and serum concentrations of erythropoietin were analyzed by ELISA (R&D Systems, Minneapolis, MN). Levels of significance were adjusted for multiple comparisons. A hemolytic index was derived by principle component analysis of reticulocyte count, aspartate aminotransferase, total bilirubin and lactate dehydrogenase. Oxygen saturation of hemoglobin was determined by pulse oximetry. Results:Interleukins-8 and 10, VEGF and erythropoietin were significantly increased in sickle cell disease patients compared with controls while RANTES was significantly decreased. Among patients with sickle cell disease, interleukins-6 and 8, interferon-γ, tumor necrosis factor-α, PDGF-bb, erythropoietin and RANTES had significant positive correlations with jet velocity in bivariate analyses. Of these, only interleukin-8 and erythropoietin correlated significantly with the hemolytic index in bivariate analyses. By logistic regression, interleukin-6 (p = 0.020) and PDGF-bb (P = 0.003) were independently associated with increased odds of elevated jet velocity while VEGF was independently associated with decreased odds (P = 0.004). These associations persisted after adjustment either for the degree of hemolysis or for hemoglobin oxygen saturation. Conclusion: Similar to observations in primary and experimental pulmonary hypertension, altered expression of interleukin-6, PDGF-bb and VEGF may be associated with the development of pulmonary hypertension in children with sickle cell disease. At least some of these effects may be additive to those of hemolysis and hypoxia. Further investigations of these pathways may be appropriate in the search for new therapeutic modalities.

Description: A urine concentrating defect is quite common in sickle cell anemia (SCA), has its onset in early childhood, and may be reversible (with transfusion) until age 10 years. BABY HUG is an NHLBI-NICHD sponsored double-blind, placebo-controlled Phase III Clinical Trial (NCT00006400) designed to assess efficacy of hydroxyurea in preventing organ damage in young children with SCA (Hb SS or SβO thalassemia); primary endpoints are spleen function and glomerular filtration rate (GFR). Two hundred thirty-three infants, recruited without regard to disease severity, underwent eligibility screening. To assess urine concentrating ability as a secondary endpoint, parents were instructed to collect and save timed urine specimens from subjects after 4 to 10 hours of fluid deprivation (NPO) overnight for osmolality (OSM) determination. More prolonged deprivation was avoided due to safety concerns. A paired serum sample for OSM, urea nitrogen (BUN), and creatinine was obtained the next morning. All specimens were analyzed in a central laboratory. The analyses included 184 infants with a urine specimen and a reported period of fluid deprivation of at least four hours; 178 had concurrent sera. Mean age was 13.0±2.7 mo (range 7.5 – 17.9 mo) and mean duration of fluid deprivation was 7.4±2.4 hr (4–13 hr). Mean serum OSM was 286±6 mOsm/kg H2O and independent of age, height, weight, or duration NPO. Urine OSM (mean 410±152, median 433, range 58–794 mOsm/kg H2O) was significantly greater than serum osmolality (p [mean serum OSM + 1 SD]); twenty-two (12.0%) had urine/serum OSM ratio 〉 2 and 54 (29.4%) had urine OSM 〉 500 mOsm/L. In addition, five infants (2.7%) were isosthenuric (urine OSM within mean serum OSM ± 1 SD) and 37 (20.1%) hyposthenuric (〉 1 SD below mean serum OSM) despite instructions to withhold fluid. Urine OSM was associated with increasing 99mTc-DTPA GFR (p=0.024) and BUN (p

Description: Considerable evidence suggests that hemolysis may be a cause of vasculopathy in sickle cell disease (SCD) and be associated with pulmonary hypertension risk in both adult and pediatric SCD patients. We recently treated an SCD adult for iron deficiency. Reticulocytes, LDH, and AST were normal or near normal before iron treatment but increased markedly with iron supplementation without a commensurate increase in the hemoglobin concentration. This and similar cases in the literature suggest that iron deficiency decreases hemolysis in SCD. To test this hypothesis we examined the association of serum ferritin concentration with hemolytic parameters and tricuspid regurgitant jet velocity (TRV) in 234 SCD children enrolled in a prospective multi-center study of the prevalence and significance of pulmonary hypertension. All studies were done in children who were outpatients and in the steady state of their disease. There were 32 children with ferritin concentrations 〈 50 ng/ml and 202 with ferritins of 50 ng/ml or higher. We chose the 〈 50 ng/ml ferritin cut off value because it would be expected to include both patients with iron deficiency whose serum ferritin concentration was in the deficient range and those whose ferritin was in the low normal range as a result of sickle-related inflammation. The table shows that patients in the lower ferritin group were older and had lower MCV, fewer transfusions, lower hemolytic markers (including a hemolytic index generated by principal component analysis of LDH, AST, total bilirubin and reticulocytes), and a trend to less severe anemia. The differences remained after adjustment for the number of blood transfusions. Comparison of children with SCD at steady state according to serum ferritin concentration. Results in mean or geometric mean and 95% confidence interval unless otherwise stated. Ferritin 4 blood Tx, N (%) 3 (10) 67 (34) 0.011 - Interleukin-6 ng/ml 0.5 (0.3–1.2) 0.6 (0.5–0.8) 0.6 0.6 TRV, m/sec 2.17 (2.05–2.29) 2.28 (2.24–2.32) 0.033 0.020 Hemoglobin, g/dl 9.7 (9.0–10.5) 9.3 (9.0–9.5) 0.20 0.16 MCV, fl 77.4 (73.0–81.8) 85.9 (84.4–87.5) 0.0001 0.0008 Reticulocytes (×103/mm3) 166 (137–202) 208 (188–229) 0.022 0.06 Tot. Bilirubin (mg/dl) 1.7 (1.3–2.2) 2.3 (2.1–2.5) 0.029 0.051 LDH (mU/ml) 342 (284–411) 386 (364–410) 0.14 0.11 AST (mU/ml) 35 (29–42) 40 (38–42) 0.09 0.046 Hemolytic index −0.6 (−1.2–0.1) 0.1 (−0.1–0.4) 0.018 0.023 Plasma interleukin-6 concentrations were not different between the two groups suggesting that the differences in TRV and hemolyis were not due to increased inflammation in the high ferritin group. To further account for the effect of multiple transfusions on ferritin level we also analyzed patients who had fewer than 5 blood transfusions in their lifetime. Twenty-six patients with 〈 5 transfusions and ferritin 〈 50 ng/ml were significantly older (13.5 vs 10.5 y, P=0.004), tended to have higher Hb level (10.1 vs 9.4 g/dl, P=0.08) and had lower TRV (2.15 vs 2.29 m/s, P=0.017), MCV (77 vs 84 fl, P=0.008), and hemolytic index (−0.8 vs −0.1, P=0.028) than 131 of their counterparts who had ferritin of 50 ng/ml or higher. These results suggest that incipient or latent iron deficiency may develop as SCD children grow to early adolescence and be associated with lower hemolytic rate and TRV without a noticeable adverse effect on their anemia.