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: Abstract 2664 Background: The six-minute walk (6MW) test is used in pediatrics in clinical practice and research to determine cardiopulmonary functional status. A change in 6MW may be affected by factors not strictly related to cardiopulmonary function, which may be different in different patient populations. In children and adolescents, age and height has been found to be a strong predictor of 6MW distance. We set out to study the effects of hematological and echocardiographic variables on 6MW distance in children with sickle cell disease (SCD) and its changes over time. Methods: We reviewed prospectively collected hematological, 6MW distance, and echocardiographic data from four hundred children with SCD (including 311 Hb SS or β0) and 69 controls (including 21 Hb AS) enrolled in PUSH (Pulmonary Hypertension and the Hypoxic Response in SCD). Subjects were evaluated at baseline and after 18–24 months, as per protocol. An un-encouraged 6MW was performed in children 5 years or older by trained personnel as per the guidelines of the American Thoracic Society. Subjects were studied at steady state, at least three weeks after any acute exacerbation of SCD. We used ANOVA for univariate analysis and stepwise linear regression for multivariate analysis. Results: Median (interquartile range) 6MW in severe SCD genotype (SS and S-β0) was 444 (399-508) and in controls was 495 meters (435-539) (P = 0.0002), while it was 461 meters (408-518) in milder SCD genotypes (P=0.2 for comparison with severe genotypes) (Table 1). In multivariate analysis, Hb, WBC and history of frequent pain episodes were significantly associated to distance of 6MW. Follow up 6MW obtained in 174 SCD subjects revealed a decline of 10% or more in distance in 22% of subjects with severe genotypes and 33% of other genotypes. The decline was more frequent in the subset of SS subjects with TRV〉2.59 (40% vs 19%). CONCLUSION: Six minute walk distance is significantly shorter in children with SCD, even as young as 5 years of age, when compared to age and race appropriate controls, indicating early compromise of exercise capacity. SS and S-β-0 genotype subjects have more impairment of exercise capacity compared to milder genotypes. Predictors of 6MW distance are similar in SCD and non SCD subjects, which validates the use of this test in this patient population. Longitudinal changes in subjects with SCD are similar, with declines in about a quarter of the subjects. Patients with SS who have an elevated TRV have the highest rate of decline in 6MW. These results validate the use of 6MW as a tool for assessing exercise capacity in children with SCD. 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: Hydroxyurea and higher hemoglobin F improve the clinical course and survival in sickle cell disease, but their roles in protecting from pulmonary hypertension are not clear. We studied 399 children and adolescents with sickle cell disease at steady state; 38% were being treated with hydroxyurea. Patients on hydroxyurea had higher hemoglobin concentration and lower values for a hemolytic component derived from 4 markers of hemolysis (P ≤ .002) but no difference in tricuspid regurgitation velocity compared with those not receiving hydroxyurea; they also had higher hemoglobin F (P 〈 .001) and erythropoietin (P = .012) levels. Hemoglobin F correlated positively with erythropoietin even after adjustment for hemoglobin concentration (P 〈 .001). Greater hemoglobin F and erythropoietin each independently predicted higher regurgitation velocity in addition to the hemolytic component (P ≤ .023). In conclusion, increase in hemoglobin F in sickle cell disease may be associated with relatively lower tissue oxygen delivery as reflected in higher erythropoietin concentration. Greater levels of erythropoietin or hemoglobin F were independently associated with higher tricuspid regurgitation velocity after adjustment for degree of hemolysis, suggesting an independent relationship of hypoxia with higher systolic pulmonary artery pressure. The hemolysis-lowering and hemoglobin F–augmenting effects of hydroxyurea may exert countervailing influences on pulmonary blood pressure in sickle cell disease.

Description: Chronic anemia and intraparenchymal sickling within the kidney lead to intravascular volume expansion and an increased glomerular filtration rate (GFR) in sickle cell anemia (SCA). An elevated GFR is considered to be an early indicator of renal damage in SCA, and the pathophysiologic changes leading to sickle nephropathy and elevated GFR likely begin at a young age. The Pediatric Hydroxyurea Phase III Clinical Trial (BABY HUG), an NHLBI-sponsored multi-center double-blinded placebo-controlled study, compares hydroxyurea versus placebo in infants with SCA, with the primary goal of determining the efficacy of hydroxyurea for the prevention of organ dysfunction in the spleen and kidney. In the Feasibility and Safety Pilot, a primary objective is to assess GFR in infants with SCA between the age of 12 and 18 months by measuring plasma clearance of 99m Tc-DTPA (diethylenetriaminepentaacetic acid) and by estimating GFR using the Schwartz equation. The DTPA GFR was determined following administration of an IV bolus of 25–50 μCi/kg of the radiotracer, with venous blood samples obtained at 1, 2, and 4 hours. GFR was also calculated using the Schwartz equation: 0.55 x body length (cm) ÷ plasma creatinine (mg/dL). For both measurements, a logarithmic transformation was applied to improve linearity between the variables, and to stabilize the variance of the transformed data. To date, 17 infants with SCA (median age 13.2 months) have had GFR measurements, with no complications occurring. The geometric mean of the GFR (± SD) as measured by DTPA plasma clearance was 112 ± 14.6 mL/min/1.73m2 (range 53–178 mL/min/1.73m2). By regression analysis, the GFR was correlated with age, with an increase of approximately 10% per month (univariate p = 0.006, multivariate p = 0.02), and this correlation could not be ascribed to other age-adjusted changes in hemoglobin concentration (p = 0.35), % fetal hemoglobin (HbF, p = 0.67), white blood cell (WBC) count (p = 0.64), or platelet count (p = 0.76). The estimated GFR calculated by the Schwartz equation was not significantly correlated with age (univariate p = 0.12), and adjustments using hemoglobin, %HbF, platelet or WBC counts did not improve the correlation. There was a modest correlation between GFR determined by DTPA and the Schwartz equation (r = 0.44; p = 0.08). These data indicate that (1) GFR measurement using DTPA plasma clearance is feasible in one year-old infants with SCA; (2) renal damage as measured by an elevated DTPA GFR appears to be present early in life and to be increasing with age; (3) preliminary evaluation of the use of the Schwartz formula indicates only a modest level of correlation with results obtained using DTPA measurements; and (4) in the BABY HUG trial, further evaluation of the efficacy of hydroxyurea in preservation of renal function will likely require DTPA GFR measurements rather than GFR estimates using the Schwartz equation.

Description: BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) replication is controlled by host intrinsic antiviral restriction factors some of which are counteracted by HIV-1 accessory proteins. We recently showed that ex vivo HIV-1 infection is suppressed in PBMCs obtained from Sickle Cell Disease (SCD) patients. The inhibition was mediated in part by SAMHD1 and NF-κB inhibitor, IkBα and triggered by ferroportin, an iron export protein. Ferroportin expression reduced intracellular iron levels and inhibited cellular CDK2 activity leading to reduction of SAMHD1 phosphorylation and increased expression of IkBα. OBJECTIVES: The study was designed to further clarify the mechanism of HIV-1 inhibition in SCD and identify additional HIV-1 restriction factors that may contribute to the restriction mechanism. METHODS: A customized array was utilized to determine the expression of restriction factors in SCD PBMCs. The shRNA-mediated knockdowns of the identified genes were used to further validate the role of these factors in HIV-1 replication in cultured and primary cells. HIV-1(IIIB strain) and VSVG-pseudotyped pNL4-3.Luc.R-E-virus was used to analyze HIV-1 replication. RESULTS: Overexpression of ferroportin in THP-1 cells led to increased expression of HO-1 and p21 and reduced phosphorylation of SAMHD1. Inhibition of HO-1 and p21 by small molecules increased HIV-1 replication in SCD PBMCs suggesting that these factors contributed to the restriction mechanism. Knocking down SAMHD1 in SCD PBMC did not fully restore HIV-1 replication pointing to additional restriction factors. Analysis of HIV-1 restriction factors in SCD PBMCs using customized array showed increased expression of APOBECs, TRIMs, CH25H, CPSF6, CTR9, EIF2ak2, IFI16, MX2, PML and RTF1 mRNAs. We next tested the effect of SCD trait on HIV-1 infection ex vivo and in vivo. PBMCs obtained from SCD trait individuals showed restricted HIV-1 infection with HIV-1(IIIB strain). To further validate these findings, we compared 9 trait (HbAS) HIV-1 infected individuals with 107 non-SCD (HbAA or HbAC) HIV-1 infected individuals from Howard University HIV clinic. HIV-1 viral load and levels of HIV-1 env and gag were significantly lower in HIV-1 infected SCD trait subjects. Expression of HO-1, p21 were increased and RNR2 expression was reduced in SCD trait PBMCs. Small molecule inhibitors of HO-1 and p21 induced HIV-1 replication in SCD trait PBMCs. CONCLUSIONS: Our findings point to HO-1 and p21 as factors that, in addition to SAMHD1 and IKBα, mediate HIV-1 restriction in SCD. In SCD trait, HO-1, p21 and RNR2 play a key role in ex vivo HIV-1 restriction. Thus HIV-1 infection is deregulated not only in SCD patients but also in SCD trait individuals. The HIV-1 restriction is mediated by iron-activated antiviral restriction factors. ACKNOWLEDGMENTS: This work was supported by NIH Research Grants (1P50HL118006, 1R01HL125005, 5G12MD007597, 1UM1AI26617 and P30AI087714). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Disclosures Nekhai: NIMHD, NIH: Research Funding; NHLBI, NIH: Research Funding; NIAID, NIH: Research Funding.

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 948 Background: Amino-terminal pro-brain type natriuretic peptide (NT-proBNP) is a widely used clinical laboratory marker of left ventricular stress, although it is also known to be elevated in adults with right ventricular stress due to pulmonary hypertension associated with sickle cell disease (SCD) and other disorders. NT-proBNP is associated with early mortality in adults with SCD. Methods: Using a standard clinical laboratory assay, we measured NT-proBNP in 346 children (median age 12; IQR 7–16 years) with SCD enrolled in the Pulmonary Hypertension and the Hypoxic Response in SCD (PUSH) study, an ongoing, longitudinal and observational multicenter study of children with sickle cell disease. In order to adjust for age, a known confounding factor of NT-proBNP in young children, we compared the characteristics of children with the top quartile of NT-proBNP for each 3-year increment of age with those of children in the remaining three quartiles for that age increment. We examined several factors alone and as part of a logistic regression model. Results: In univariate analyses, high expression of NT-proBNP was associated with lower hemoglobin levels (median 83 vs. 87 gm/L, p=0.0006). The high NT-proBNP group also had a higher hemolytic component, a principal component-derived index of the common properties of the hemolytic markers reticulocyte count, serum lactate dehydrogenase, aspartate aminotransferase and bilirubin (median 0.5 vs. 0.7 relative units, p=0.021). The high NT-proBNP group had a higher prevalence of high left ventricular filling pressure, as indicated by the mitral inflow E wave to tissue Doppler E wave (E/Etdi) above 9.22 (13.1% vs. 2.9%, p9.22 were independently associated with high NT-proBNP levels. Conclusions: After adjustment for age in children with SCD, upper quartile NTproBNP is independently associated with severity of anemia and echocardiographic markers of left ventricular size and diastolic filling pressure. At this age, they have not yet developed the association observed prominently in SCD adults between NT-proBNP and elevated pulmonary arterial pressures. Our data lends support to childhood being considered as a pre-symptomatic phase of pulmonary hypertension; which could be targeted for clinical trials of preventative strategies to prevent adult onset of pulmonary vascular changes associated with higher NT-proBNP and higher TRV. Hydroxyurea therapy is associated in our results from children with SCD with lower NT-proBNP, making it an attractive candidate for a trial in children with SCD to prevent pulmonary hypertension in adulthood. NT-proBNP in childhood SCD remains a marker of left ventricular measures, identifying this additional feature of cardiopulmonary risk. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1647 Clinical B12 (cobalamin) deficiency occurs in sickle cell disease (SCD) but it is uncommon (NEJM 2003;348:2204). Several publications report lower serum B12 concentrations in SCD subjects than in controls (Acta Haematol 1984;71:299; J Intern Med 1995;237:551; J Nat Med Assoc 2006;98:352). However, other studies do not show these differences (Am J Hematol 2004;76:114; South Med J 2004;97:149; J Am Coll Nutr 2000;19:608). Serum transcobalamin concentrations are high in SCD (Scand J Haematol 1983;30:135; Acat Haematol 1989;81:117) so that one would expect higher than normal B12 levels in these patients. We measured serum B12 concentrations in 467 children and adolescents with SCD and in 69 control subjects matched for age, sex, and ethnicity. All subjects were enrolled in the PUSH protocol, a multicenter study for determining the prevalence and significance of pulmonary hypertension in children with SCD. The median serum B12 concentration in SCD was lower than in controls (960 vs. 1094 pg/ml, p=0.003). No patient had a serum B12 value lower than 227 pg/ml (3 SD below the mean of control subjects) and sickle genotype did not affect B12 concentration. Univariate analysis in children with the Hb SS genotype (N=343) showed that age, body mass index, serum creatinine (all three p=0.0001), and red cell MCV (p=0.025) were each inversely correlated with B12 levels, and this suggested the possibility that as children grow, their B12 requirements increase. In SS children higher leukocyte counts, serum AST, and serum ALT were all associated with higher B12 levels (p=0.005, p=0.0001, and 0.011, respectively), while blood Hb concentration, a-thalassemia status, Hb F %, platelet count, and hemolytic parameters were unrelated to serum B12. The median B12 level in SS children (941 pg/ml) remained significantly lower than that of controls even after adjustment for age and WBC. Children with Hb SS who were taking hydroxyurea had significantly lower median B12 levels (845 pg/ml, N=141) than SS children not on this drug (992 pg/ml, N=200 p=0.027) (Figure) but after adjustment for age and WBC this difference was no longer significant (p=0.5). These results suggest that (a) children with SCD have lower, though still normal, serum B12 concentrations than healthy subjects, (b) this difference is not explained by SCD leukocytosis so that it could represent increased B12 requirements, and (c) hydroxyurea treatment reduces B12 levels even further probably as a result of its lowering effect on leukocyte count. However, because hydroxyurea was not given in a randomized manner, we cannot exclude the confounding effect of age on B12 level in hydroxurea treated subjects. Since we and others (Acat Haematol 1989;81:117) show that in SCD serum B12 decreases with age, as is the case also in non-SCD individuals (Am J Clin Nutr 1997;66:741), it would seem prudent to monitor cobalamin levels in SCD adults, particularly in older subjects and in those taking hydroxyurea. Disclosures: No relevant conflicts of interest to declare.