Hydroxyurea to prevent brain injury in children with sickle cell disease (HU Prevent)-A randomized, placebo-controlled phase II feasibility/pilot study.

Central nervous system (CNS) injury is common in sickle cell disease (SCD) and occurs early in life. Hydroxyurea is safe and efficacious for treatment of SCD, but high-quality evidence from randomized trials to estimate its neuroprotective effect is scant. HU Prevent was a randomized (1:1), double-blind, phase II feasibility/pilot trial of dose-escalated hydroxyurea vs. placebo for the primary prevention of CNS injury in children with HbSS or HbS-ß0-thalassemia subtypes of SCD age 12-48 months with normal neurological examination, MRI of the brain, and cerebral blood flow velocity. We hypothesized that hydroxyurea would reduce by 50% the incidence of CNS injury. Two outcomes were compared: primary-a composite of silent cerebral infarction, elevated cerebral blood flow velocity, transient ischemic attack, or stroke; secondary-a weighted score estimating the risk of suffering the consequences of stroke (the Stroke Consequences Risk Score-SCRS), based on the same outcome events. Six participants were randomized to each group. One participant in the hydroxyurea group had a primary outcome vs. four in the placebo group (incidence rate ratio [90% CI] 0.216 [0.009, 1.66], p = .2914) (~80% reduction in the hydroxyurea group). The mean SCRS score was 0.078 (SD 0.174) in the hydroxyurea group, 0.312 (SD 0.174) in the placebo group, p = .072, below the p-value of .10 often used to justify subsequent phase III investigations. Serious adverse events related to study procedures occurred in 3/41 MRIs performed, all related to sedation. These results suggest that hydroxyurea may have profound neuroprotective effect in children with SCD and support a definitive phase III study to encourage the early use of hydroxyurea in all infants with SCD.

Proteomic discovery in sickle cell disease: Elevated neurogranin levels in children with sickle cell disease.

PURPOSE: Sickle cell disease (SCD) is an inherited hemoglobinopathy that causes stroke and silent cerebral infarct (SCI). Our aim was to identify markers of brain injury in SCD. EXPERIMENTAL DESIGN: Plasma proteomes were analyzed using a sequential separation approach of hemoglobin (Hb) and top abundant plasma protein depletion, followed by reverse phase separation of intact proteins, trypsin digestion, and tandem mass spectrometry. We compared plasma proteomes of children with SCD with and without SCI in the Silent Cerebral Infarct Multi-Center Clinical Trial (SIT Trial) to age-matched, healthy non-SCD controls. RESULTS: From the SCD group, 1172 proteins were identified. Twenty-five percent (289/1172) were solely in the SCI group. Twenty-five proteins with enriched expression in the human brain were identified in the SCD group. Neurogranin (NRGN) was the most abundant brain-enriched protein in plasma of children with SCD. Using a NRGN sandwich immunoassay and SIT Trial samples, median NRGN levels were higher at study entry in children with SCD (0.28 ng/mL, N = 100) compared to control participants (0.12 ng/mL, N = 25, p < 0.0004). CONCLUSIONS AND CLINICAL RELEVANCE: NRGN levels are elevated in children with SCD. NRGN and other brain-enriched plasma proteins identified in plasma of children with SCD may provide biochemical evidence of neurological injury.

Brain-derived neurotrophic factor levels in pediatric sickle cell disease.

BACKGROUND: Children with sickle cell disease (SCD) have an increased risk of neurological complications, particularly stroke and silent cerebral infarction (SCI). Brain-derived neurotrophic factor (BDNF) is a nerve growth factor associated with neuronal survival, synaptic plasticity, elevated transcranial Doppler (TCD) velocities and increased risk of stroke in patients with SCD. The objective of this study was to analyze plasma BDNF protein levels in children with SCD participating in the Silent Cerebral Infarct Transfusion Multi-Center Clinical Trial (SIT Trial), comparing plasma samples of children with SCD and SCI to plasma samples from children with SCD without SCI, as well as healthy pediatric control participants. PROCEDURE: Entry, exit, and longitudinal blood samples were collected from 190 SIT Trial participants with SCD and healthy pediatric controls over time. BDNF levels were measured by enzyme-linked immunosorbent assay. Sample collection was not optimized for measurements of BDNF, but factors affecting BDNF levels were accounted for in analyses. RESULTS: BDNF levels were significantly higher in children with SCD in comparison to healthy pediatric control subjects. BDNF levels significantly increased over time in SCD participants. BDNF levels did not show any significant associations with the presence or absence of SCI or new/progressive SCI/stroke or TCD velocities. CONCLUSIONS: Plasma BDNF levels are elevated and increase over time in children with SCD. Additional studies with more longitudinal samples are needed to address the reasons for those increased levels.

Identification of Aptamers That Bind to Sickle Hemoglobin and Inhibit Its Polymerization.

The pathophysiology of sickle cell disease (SCD) is dependent on the polymerization of deoxygenated sickle hemoglobin (HbS), leading to erythrocyte deformation (sickling) and vaso-occlusion within the microvasculature. Following deoxygenation, there is a delay time before polymerization is initiated, during which nucleation of HbS monomers occurs. An agent with the ability to extend this delay time or slow polymerization would therefore hold a therapeutic, possibly curative, potential. We used the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method to screen for HbS-binding RNA aptamers modified with nuclease-resistant 2'-fluoropyrimidines. Polymerization assays were employed to identify aptamers with polymerization-inhibitory properties. Two noncompeting aptamers, DE3A and OX3B, were found to bind hemoglobin, significantly increase the delay time, and reduce the rate of polymerization of HbS. These modifiable, nuclease-resistant aptamers are potential new therapeutic agents for SCD.

Proteomic and biomarker studies and neurological complications of pediatric sickle cell disease.

Biomarker analysis and proteomic discovery in pediatric sickle cell disease has the potential to lead to important discoveries and improve care. The aim of this review article is to describe proteomic and biomarker articles involving neurological and developmental complications in this population. A systematic review was conducted to identify relevant research publications. Articles were selected for children under the age of 21 years with the most common subtypes of sickle cell disease. Included articles focused on growth factors (platelet-derived growth factor), intra and extracellular brain proteins (glial fibrillary acidic protein, brain-derived neurotrophic factor), and inflammatory and coagulation markers (interleukin-1ß, l-selectin, thrombospondin-1, erythrocyte, and platelet-derived microparticles). Positive findings include increases in plasma brain-derived neurotrophic factor and platelet-derived growth factor with elevated transcranial Dopplers velocities, increases in platelet-derived growth factor isoform AA with overt stroke, and increases in glial fibrillary acidic protein with acute brain injury. These promising potential neuro-biomarkers provide insight into pathophysiologic processes and clinical events, but their clinical utility is yet to be established. Additional proteomics research is needed, including broad-based proteomic discovery of plasma constituents and blood cell proteins, as well as urine and cerebrospinal fluid components, before, during and after neurological and developmental complications.

Genome-wide meta-analysis of systolic blood pressure in children with sickle cell disease.

In pediatric sickle cell disease (SCD) patients, it has been reported that higher systolic blood pressure (SBP) is associated with increased risk of a silent cerebral infarction (SCI). SCI is a major cause of neurologic morbidity in children with SCD, and blood pressure is a potential modulator of clinical manifestations of SCD; however, the risk factors underlying these complications are not well characterized. The aim of this study was to identify genetic variants that influence SBP in an African American population in the setting of SCD, and explore the use of SBP as an endo-phenotype for SCI. We conducted a genome-wide meta-analysis for SBP using two SCD cohorts, as well as a candidate screen based on published SBP loci. A total of 1,617 patients were analyzed, and while no SNP reached genome-wide significance (P-value<5.0 x 10(-8)), a number of suggestive candidate loci were identified. The most significant SNP, rs7952106 (P-value=8.57 x 10(-7)), was in the DRD2 locus on chromosome 11. In a gene-based association analysis, MIR4301 (micro-RNA4301), which resides in an intron of DRD2, was the most significant gene (P-value=5.2 x 10(-5)). Examining 27 of the previously reported SBP associated SNPs, 4 SNPs were nominally significant. A genetic risk score was constructed to assess the aggregated genetic effect of the published SBP variants, demonstrating a significant association (P=0.05). In addition, we also assessed whether these variants are associated with SCI, validating the use of SBP as an endo-phenotype for SCI. Three SNPs were nominally associated, and only rs2357790 (5' CACNB2) was significant for both SBP and SCI. None of these SNPs retained significance after Bonferroni correction. Taken together, our results suggest the importance of DRD2 genetic variation in the modulation of SBP, and extend the aggregated importance of previously reported SNPs in the modulation of SBP in an African American cohort, more specifically in children with SCD.

Acute chest syndrome is associated with single nucleotide polymorphism-defined beta globin cluster haplotype in children with sickle cell anaemia.

Genetic diversity at the human ß-globin locus has been implicated as a modifier of sickle cell anaemia (SCA) severity. However, haplotypes defined by restriction fragment length polymorphism sites across the ß-globin locus have not been consistently associated with clinical phenotypes. To define the genetic structure at the ß-globin locus more thoroughly, we performed high-density single nucleotide polymorphism (SNP) mapping in 820 children who were homozygous for the sickle cell mutation (HbSS). Genotyping results revealed very high linkage disequilibrium across a large region spanning the locus control region and the HBB (ß-globin gene) cluster. We identified three predominant haplotypes accounting for 96% of the ß(S) -carrying chromosomes in this population that could be distinguished using a minimal set of common SNPs. Consistent with previous studies, fetal haemoglobin level was significantly associated with ß(S) -haplotypes. After controlling for covariates, an association was detected between haplotype and rate of hospitalization for acute chest syndrome (ACS) (incidence rate ratio 0·51, 95% confidence interval 0·29-0·89) but not incidence rate of vaso-occlusive pain or presence of silent cerebral infarct (SCI). Our results suggest that these SNP-defined ß(S) -haplotypes may be associated with ACS, but not pain or SCI in a study population of children with SCA.

Association between baseline fetal hemoglobin levels and incidence of severe vaso-occlusive pain episodes in children with sickle cell anemia.

The ameliorating effect of high fetal hemoglobin (HbF) levels on the incidence of pain episodes in sickle cell anemia (SCA) is well-known; however, in children this relationship is less clearly established. We hypothesized that higher HbF levels in children with SCA are associated with fewer severe pain episodes. A meta-analysis of data from the Silent Infarct Transfusion Trial (n = 456) and the Cooperative Study of Sickle Cell Disease (n = 764), demonstrated that baseline HbF levels were associated with the incidence of severe pain, commonly defined across studies as an event requiring hospitalization (P-value = 0.02).

Magnetic resonance angiography-defined intracranial vasculopathy is associated with silent cerebral infarcts and glucose-6-phosphate dehydrogenase mutation in children with sickle cell anaemia.

Silent cerebral infarct (SCI) is the most commonly recognized cause of neurological injury in sickle cell anaemia (SCA). We tested the hypothesis that magnetic resonance angiography (MRA)-defined vasculopathy is associated with SCI. Furthermore, we examined genetic variations in glucose-6-phosphate dehydrogenase (G6PD) and HBA (α-globin) genes to determine their association with intracranial vasculopathy in children with SCA. Magnetic resonance imaging (MRI) of the brain and MRA of the cerebral vasculature were available in 516 paediatric patients with SCA, enrolled in the Silent Infarct Transfusion (SIT) Trial. All patients were screened for G6PD mutations and HBA deletions. SCI were present in 41·5% (214 of 516) of SIT Trial children. The frequency of intracranial vasculopathy with and without SCI was 15·9% and 6·3%, respectively (P < 0·001). Using a multivariable logistic regression model, only the presence of a SCI was associated with increased odds of vasculopathy (P = 0·0007, odds ratio (OR) 2·84; 95% Confidence Interval (CI) = 1·55-5·21). Among male children with SCA, G6PD status was associated with vasculopathy (P = 0·04, OR 2·78; 95% CI = 1·04-7·42), while no significant association was noted for HBA deletions. Intracranial vasculopathy was observed in a minority of children with SCA, and when present, was associated with G6PD status in males and SCI.

Heme oxygenase-1 gene promoter polymorphism is associated with reduced incidence of acute chest syndrome among children with sickle cell disease.

Sickle cell disease is a common hemolytic disorder with a broad range of complications, including vaso-occlusive episodes, acute chest syndrome (ACS), pain, and stroke. Heme oxygenase-1 (gene HMOX1; protein HO-1) is the inducible, rate-limiting enzyme in the catabolism of heme and might attenuate the severity of outcomes from vaso-occlusive and hemolytic crises. A (GT)(n) dinucleotide repeat located in the promoter region of the HMOX1 gene is highly polymorphic, with long repeat lengths linked to decreased activity and inducibility. We examined this polymorphism to test the hypothesis that short alleles are associated with a decreased risk of adverse outcomes (hospitalization for pain or ACS) among a cohort of 942 children with sickle cell disease. Allele lengths varied from 13 to 45 repeats and showed a trimodal distribution. Compared with children with longer allele lengths, children with 2 shorter alleles (4%; ≤ 25 repeats) had lower rates of hospitalization for ACS (incidence rate ratio 0.28, 95% confidence interval, 0.10-0.81), after adjusting for sex, age, asthma, percentage of fetal hemoglobin, and α-globin gene deletion. No relationship was identified between allele lengths and pain rate. We provide evidence that genetic variation in HMOX1 is associated with decreased rates of hospitalization for ACS, but not pain. This study is registered at www.clinicaltrials.gov as #NCT00072761.

A genome-wide association study of total bilirubin and cholelithiasis risk in sickle cell anemia.

Serum bilirubin levels have been associated with polymorphisms in the UGT1A1 promoter in normal populations and in patients with hemolytic anemias, including sickle cell anemia. When hemolysis occurs circulating heme increases, leading to elevated bilirubin levels and an increased incidence of cholelithiasis. We performed the first genome-wide association study (GWAS) of bilirubin levels and cholelithiasis risk in a discovery cohort of 1,117 sickle cell anemia patients. We found 15 single nucleotide polymorphisms (SNPs) associated with total bilirubin levels at the genome-wide significance level (p value <5 × 10(-8)). SNPs in UGT1A1, UGT1A3, UGT1A6, UGT1A8 and UGT1A10, different isoforms within the UGT1A locus, were identified (most significant rs887829, p = 9.08 × 10(-25)). All of these associations were validated in 4 independent sets of sickle cell anemia patients. We tested the association of the 15 SNPs with cholelithiasis in the discovery cohort and found a significant association (most significant p value 1.15 × 10(-4)). These results confirm that the UGT1A region is the major regulator of bilirubin metabolism in African Americans with sickle cell anemia, similar to what is observed in other ethnicities.

Plasma glial fibrillary acidic protein levels in children with sickle cell disease.

To determine if glial fibrillary acidic protein (GFAP) is associated with brain injury in children with sickle cell disease (SCD), we measured plasma GFAP among cross-sectional groups of unselected children with SCD, subsets of children with SCD and normal brain MRI or MRI evidence of cerebral infarct, healthy pediatric controls, and adults with brain injury. Children with SCD had higher plasma GFAP than healthy pediatric controls (mean concentrations 0.14 ± 0.37 vs. 0.07 ± 0.08 ng/mL; P 5 0.003); also, 16.0% (16/100) of children with SCD and cerebral infarct had GFAP elevations above the 95th percentile of healthy pediatric controls (P 5 0.04). Although not statistically significant, children with SCD and cerebral infarct had more elevated GFAP levels than with SCD and no infarct (16/100, 16.0% vs. 14/168, 8.3%; P 5 0.07). Children with SCD and acute brain ischemia had a higher proportion of elevated GFAP than SCD children with normal MRI (3/6, 50% vs.8.3%; P 5 0.01). GFAP was associated with elevated systolic blood pressure in the preceding year and correlated positively with white blood cell count and negatively with age and performance IQ. Plasma GFAP is elevated among children with SCD and may be associated with subclinical brain injury.

Elevation of IgE in children with sickle cell disease is associated with doctor diagnosis of asthma and increased morbidity.

BACKGROUND: A doctor's diagnosis of asthma is associated with increased morbidity (pain and acute chest syndrome [ACS]) among children with sickle cell disease (SCD). An association between IgE levels and asthma and morbidity has not been investigated in children with SCD. OBJECTIVE: We tested the hypothesis that elevated total and allergen-specific IgE levels are associated with asthma and SCD morbidity in children with SCD. METHODS: A cross-sectional study of children with SCD who participated in the Silent Cerebral Infarct Trial was conducted. Logistic regression and negative binomial regression were used to investigate potential associations of total and allergen-specific IgE levels with asthma diagnosis and SCD morbidity, both confirmed by medical record review. Elevation of total IgE level was defined as age-adjusted and sex-adjusted IgE level exceeding the 90th percentile compared with a nonatopic reference population. IgE antibody positivity to Alternaria alternata (mold), Blattellagermanica (cockroach), and Dermatophagoides pteronyssinus (dust mite) was assessed by ImmunoCAP analysis. RESULTS: Children with SCD (140 with asthma; 381 without asthma) were evaluated. Elevations in total IgE level (P = .04) and IgE antibody specific for Alternaria alternata (P = .0003), Blattella germanica (P = .008), and Dermatophagoides pteronyssinus (P = .01) were associated with asthma. ACS (P = .048) but not pain (P = .20) was associated with total IgE level, but neither was associated with specific IgE levels. CONCLUSION: Significantly increased levels of total and allergen-specific IgE levels are associated with asthma in SCD. High IgE levels are a risk factor for ACS but not pain rates.

Genome-wide association study identifies genetic variants influencing F-cell levels in sickle-cell patients.

Fetal hemoglobin (HbF) level has emerged as an important prognostic factor in sickle-cell disease (SCD) and can be measured by the proportion of HbF-containing erythrocytes (F-cells). Recently, BCL11A (zinc-finger protein) was identified as a regulator of HbF, and the strongest association signals were observed either directly for rs766432 or for correlated single-nucleotide polymorphisms (SNPs). To identify additional independently associated genetic variants, we performed a genome-wide association study (GWAS) on the proportion of F-cells in individuals of African ancestry with SCD from the Silent Infarct Transfusion (SIT) Trial cohort. Our study not only confirms the association of rs766432 (P-value <3.32 × 10(-13)), but also identifies an independent novel intronic SNP, rs7606173, associated with F-cells (P-value <1.81 × 10(-15)). The F-cell variances explained independently by these two SNPs are ∼13% (rs7606173) and ∼11% (rs766432), whereas, together they explain ∼16%. Additionally, in men, we identify a novel locus on chromosome 17, glucagon-like peptide-2 receptor (GLP2R), associated with F-cell regulation (rs12103880; P-value <3.41 × 10(-8)). GLP2R encodes a G protein-coupled receptor and involved in proliferative and anti-apoptotic cellular responses. These findings highlight the importance of denser genetic screens and suggest further exploration of the BCL11A and GLP2R loci to gain additional insight into HbF/F-cell regulation.

Hemoglobin depletion from plasma: considerations for proteomic discovery in sickle cell disease and other hemolytic processes.

PURPOSE: Hemoglobin (Hb) depletion with nickel affinity chromatography has been shown to increase the number of proteins identified in proteomic studies of erythrocytes, but limited data exist on the application of this technique in depletion of Hb from plasma or serum required for clinical biomarker studies. The aim of this study was to explore the potential of using nickel-beads for Hb depletion of plasma. EXPERIMENTAL DESIGN: Nickel­nitrilotriacetic acid (Ni­NTA) affinity chromatography was used to deplete Hb from hemolyzed plasma samples obtained from children with sickle cell disease (SCD, n=7) and normal human plasma (n=4). Ni­NTA-bound proteins were analyzed by one-dimensional GE, followed by in-gel digestion for characterization using an LTQ-Orbitrap hybrid mass spectrometer. In addition, the loss of two non-Hb-related plasma proteins, thrombospondin1 and L-selectin, by Ni­NTA was determined by ELISA (SCD n=6, non-SCD controls n=2). RESULTS: Ni­NTA resulted in an average 60% decrease in plasma protein concentration, which was not hemolysis dependent. Specifically, Hb (7 peptides) and the top three proteins, -2-macroglobulin (75 peptides), apolipoprotein B-100 (73 peptides), and albumin (42 peptides) were Ni­NTA bound. In addition, using an ELISA assay two non-Hb-associated plasma proteins thrombospondin1 and L-selectin were decreased by Ni-NTA. CONCLUSIONS AND CLINICAL RELEVANCE: Hb depletion with Ni­NTA is effective for Hb removal but is not specific. There is a potential for deleterious depletion of potential biomarkers that may limit the applicability of this method. Consideration of alternate methods of Hb depletion for clinical proteomics may be warranted.

The role of peroxisomal ABC transporters in the mouse adrenal gland: the loss of Abcd2 (ALDR), Not Abcd1 (ALD), causes oxidative damage.

X-linked adreno-leukodystrophy is a progressive, systemic peroxisomal disorder that primarily affects the adrenal cortex, as well as myelin and axons of the central nervous system. Marked phenotypic heterogeneity does not correlate with disease-causing mutations in ABCD1, which encodes a peroxisomal membrane protein that is a member of the ABC transmembrane transporter proteins. The precise physiological functions of ABCD1 and ABCD2, a closely related peroxisomal membrane half-transporter, are unknown. The abcd1 knockout mouse does not develop the inflammatory demyelination so typical and devastating in adreno-leukodystrophy, but it does display the same lamellae and lipid profiles in adrenocortical cells under the electron microscope as the human patients. The adrenocortical cells in the mouse also exhibit immunohistochemical evidence of oxidative stress at 12 weeks but no evidence of oxidative damage. To better understand the pathogenesis of this complex disease, we evaluate the adrenal lesion of the abcd1 knockout mouse as a function of normal aging, dietary or therapeutic manipulations, and abcd genotype. The loss of abcd2 causes oxidative stress in the adrenal at 12 weeks, as judged by increased immunoreactivity for the mitochondrial manganese superoxide dismutase, in both the inner cortex and medulla. The loss of abcd2 (n=20), but not abcd1 (n=27), results in the spontaneous and premature deposition of ceroid, a known end-product of oxidative damage, predominantly in adrenal medullary cells. These data indicate that the loss of abcd2 results in greater oxidative stress in murine adrenal cells than the loss of abcd1, providing a clue to its cellular function. We also find that the adrenocortical lesion of the abcd1 knockout mouse does not produce functional impairment at ten to nineteen months or overt hypocortisolism at any age, nor does it progress histologically; these and other data align this mouse model closer to human female heterozygotes than to male ALD or AMN hemizygotes.