Genome-wide association study of early ischaemic stroke risk in Brazilian individuals with sickle cell disease implicates ADAMTS2 and CDK18 and uncovers novel loci.

Ischaemic stroke is a common complication of sickle cell disease (SCD) and without intervention can affect 11% of children with SCD before the age of 20. Within the Trans-Omics for Precision Medicine (TOPMed), a genome-wide association study (GWAS) of ischaemic stroke was performed on 1333 individuals with SCD from Brazil (178 cases, 1155 controls). Via a novel Cox proportional-hazards analysis, we searched for variants associated with ischaemic stroke occurring at younger ages. Variants at genome-wide significance (p < 5 × 10-8 ) include two near genes previously linked to non-SCD early-onset stroke (<65 years): ADAMTS2 (rs147625068, p = 3.70 × 10-9 ) and CDK18 (rs12144136, p = 2.38 × 10-9 ). Meta-analysis, which included the independent SCD cohorts Walk-PHaSST and PUSH, exhibited consistent association for variants rs1209987 near gene TBC1D32 (p = 3.36 × 10-10 ), rs188599171 near CUX1 (p = 5.89 × 10-11 ), rs77900855 near BTG1 (p = 4.66 × 10-8 ), and rs141674494 near VPS13C (1.68 × 10-9 ). Findings from this study support a multivariant model of early ischaemic stroke risk and possibly a shared genetic architecture between SCD individuals and non-SCD individuals younger than 65 years.

Decreased parasite burden and altered host response in children with sickle cell anemia and severe anemia with malaria.

Plasmodium falciparum malaria causes morbidity and mortality in African children with sickle cell anemia (SCA), but comparisons of host responses to P falciparum between children with SCA (homozygous sickle cell disease/hemoglobin SS [HbSS]) and normal hemoglobin genotype/hemoglobin AA (HbAA) are limited. We assessed parasite biomass and plasma markers of inflammation and endothelial activation in children with HbAA (n = 208) or HbSS (n = 22) who presented with severe anemia and P falciparum parasitemia to Mulago Hospital in Kampala, Uganda. Genotyping was performed at study completion. No child had known SCA at enrollment. Children with HbSS did not differ from children with HbAA in peripheral parasite density, but had significantly lower sequestered parasite biomass. Children with HbSS had greater leukocytosis but significantly lower concentrations of several plasma inflammatory cytokines, including tumor necrosis factor α (TNF-α). In contrast, children with HbSS had threefold greater concentrations of angiopoietin-2 (Angpt-2), a marker of endothelial dysregulation associated with mortality in severe malaria. Lower TNF-α concentrations were associated with increased risk of postdischarge mortality or readmission, whereas higher Angpt-2 concentrations were associated with increased risk of recurrent clinical malaria. Children with SCA have decreased parasite sequestration and inflammation but increased endothelial dysregulation during severe anemia with P falciparum parasitemia, which may ameliorate acute infectious complications but predispose to harmful long-term sequelae.

Hydroxycarbamide treatment reduces transcranial Doppler velocity in the absence of transfusion support in children with sickle cell anaemia, elevated transcranial Doppler velocity, and cerebral vasculopathy: the EXTEND trial.

EXpanding Treatment for Existing Neurological Disease (EXTEND) investigated whether hydroxycarbamide lowers transcranial Doppler (TCD) velocities in Jamaican children with sickle cell anaemia (SCA) and elevated TCD velocity with or without previous stroke. Forty-three children (age 2-17 years) with baseline maximum time-averaged mean velocity (TAMV) ≥ 170 cm/s were stratified into three risk categories based on treatment status and stroke history: Group 1 (no history of stroke, on hydroxycarbamide, n = 12); and Groups 2 (no stroke, no hydroxycarbamide, n = 21) and 3 (previous stroke, no hydroxycarbamide, n = 10). Open-label hydroxycarbamide at 20 mg/kg/day was commenced, with escalation to maximum tolerated dose (MTD) based on mild marrow suppression (average dose 25·4 ± 4·5 mg/kg/day). TCD was performed every six months with brain magnetic resonance imaging (MRI)/magnetic resonance angiography (MRA) at baseline and after 18-months of hydroxycarbamide. The maximum TAMV decreased significantly compared to baseline (24 ± 30 cm/s, P < 0·0001), with similar declines in all groups. Clinical stroke occurred in five children, one in Group 1, none in Group 2, and four in Group 3, P = 0·0032, comparing group incidence rates. Brain MRI/MRA was stable in children without clinical stroke. EXTEND documents the feasibility and benefits of hydroxycarbamide at MTD to lower TCD velocities and reduce stroke risk in children with SCA and no history of primary stroke in low-resource settings without transfusion management.

Hydroxyurea Exposure in Lactation: a Pharmacokinetics Study (HELPS).

Lactation is contraindicated for women with sickle cell anemia receiving hydroxyurea therapy, despite sparse pharmacokinetics data. In 16 women who were lactating volunteers, we documented hydroxyurea transferred into breastmilk with a relative infant dosage of 3.4%, which is below the recommended 5%-10% safety threshold. Breastfeeding should be permitted for women taking daily oral hydroxyurea.

Transcranial Doppler velocity among Jamaican children with sickle cell anaemia: determining the significance of haematological values and nutrition.

This study investigated the association of nutritional and haematological variables with maximum time-averaged mean velocity (TAMV) measured by transcranial Doppler (TCD) velocity and the agreement of classification between two protocols. TCD categories included: normal (<170 cm/s), conditional (170-199 cm/s) and abnormal (≥200 cm/s) based on TAMV in distal internal carotid artery (dICA), middle cerebral artery (MCA), internal carotid bifurcation, anterior and posterior cerebral arteries. Of 358 children with sickle cell anaemia (SCA) examined, the mean age (±standard deviation) was 7·4 ± 2·7 years; 13·1% and 6·7% had conditional and abnormal velocities, respectively. Children with abnormal TCD velocities had higher prevalence of prior stroke (P = 0·006). Increased TAMV was associated with younger age (P = 0·001), lower weight (P = 0·001), height (P = 0·007) and oxygen saturation (P = 0·005). There was no association of TAMV with height-age or body mass index (BMI) z-scores. Adjusting for gender, BMI z-score, age, previous stroke and oxygen saturation, mean corpuscular volume (P = 0·005) and reticulocyte count (P = 0·013) were positively associated with TAMV, while haemoglobin concentration (P = 0·009) was negatively associated. There was good agreement [99%; weighted Kappa 0·98 (95% confidence interval 0·89-1), P = 0·0001] in TCD classification using data from five vessels versus two vessels (dICA and MCA). Haematological variables, rather than nutritional status, may be useful markers that identify high-risk children with SCA.

Stroke Avoidance for Children in REpública Dominicana (SACRED): Protocol for a Prospective Study of Stroke Risk and Hydroxyurea Treatment in Sickle Cell Anemia.

BACKGROUND: In the Dominican Republic, where the burden of sickle cell anemia (SCA) is high, many children lack access to routine screening and preventative care. Children with SCA are at risk for stroke, an event that leads to significant morbidity and mortality. In the United States, screening via transcranial Doppler (TCD) identifies children with SCA at highest stroke risk, allowing early intervention with blood transfusions. The need for indefinite transfusions for primary stroke prevention limits their practicality in limited-resource countries. Hydroxyurea has been shown to lower TCD velocities and to prevent conversion from conditional (170 to 199 cm/sec) to abnormal (greater than or equal to 200 cm/sec) velocities. In resource-limited settings, implementation of a TCD screening program, coupled with hydroxyurea therapy, could reduce the burden of SCA and stroke. OBJECTIVE: The aims of the Stroke Avoidance for Children in REpública Dominicana (SACRED) trial are (1) to screen children with SCA for stroke risk using TCD and to determine the prevalence of elevated velocities in a cross-sectional sample; (2) to identify clinical and laboratory correlates of elevated velocities; and (3) to obtain longitudinal data on the natural history of TCD velocities and to measure therapeutic effects of hydroxyurea. METHODS: This prospective trial, designed and conducted by Cincinnati Children's Hospital Medical Center (CCHMC) and Hospital Infantil Robert Reid Cabral (HIRRC) with Centro de Obstetricia y Ginecología, includes a baseline cross-sectional epidemiological survey of the distribution of TCD velocities across a large cohort of children with SCA in the Dominican Republic. Children with conditional velocities are eligible to begin protocol-directed hydroxyurea if laboratory criteria are met. The treatment schedule begins with a fixed-dose of approximately 20 mg/kg/day for 6 months, after which it escalates to maximum tolerated dose (MTD). All participants undergo longitudinal annual TCD evaluation, while those on hydroxyurea have semi-annual evaluations during the 3-year study period. Data are collected using an Internet-based Research Electronic Data Capture (REDCap) system with forms translated into Spanish; both remote and on-site monitoring are used. RESULTS: To date, 122 children with SCA have enrolled in SACRED including 85 (69.7%, 85/122) with normal, 29 (23.8%, 29/122) with conditional, 5 (4.1%, 5/122) with abnormal, and 3 (2.5%, 3/122) with inadequate TCD velocities. Of the 29 children with conditional TCD velocities, 17 (59%, 17/29) have initiated hydroxyurea per protocol, with plans for escalation to MTD. CONCLUSIONS: The SACRED trial will provide novel epidemiologic data about the prevalence of children with SCA and increased stroke risk in the Dominican Republic. The study also includes an investigation of the impact of hydroxyurea at MTD on elevated TCD velocities, as well as clinical and laboratory parameters. The design and implementation of SACRED reflect a successful international institutional partnership, one that features local capacity building and training in research methods and clinical care. The trial's results have important implications for screening and prevention of primary stroke in children with SCA living in resource-limited settings. TRIAL REGISTRATION: ClinicalTrials.gov NCT02769845; https://www.clinicaltrials.gov/ct2/show/NCT02769845 (Archived by WebCite at http://www.webcitation.org/6qf6n0Egh).

Whole-exome sequencing for RH genotyping and alloimmunization risk in children with sickle cell anemia.

RH genes are highly polymorphic and encode the most complex of the 35 human blood group systems. This genetic diversity contributes to Rh alloimmunization in patients with sickle cell anemia (SCA) and is not avoided by serologic Rh-matched red cell transfusions. Standard serologic testing does not distinguish variant Rh antigens. Single nucleotide polymorphism (SNP)-based DNA arrays detect many RHD and RHCE variants, but the number of alleles tested is limited. We explored a next-generation sequencing (NGS) approach using whole-exome sequencing (WES) in 27 Rh alloimmunized and 27 matched non-alloimmunized patients with SCA who received chronic red cell transfusions and were enrolled in a multicenter study. We demonstrate that WES provides a comprehensive RH genotype, identifies SNPs not interrogated by DNA array, and accurately determines RHD zygosity. Among this multicenter cohort, we demonstrate an association between an altered RH genotype and Rh alloimmunization: 52% of Rh immunized vs 19% of non-immunized patients expressed variant Rh without co-expression of the conventional protein. Our findings suggest that RH allele variation in patients with SCA is clinically relevant, and NGS technology can offer a comprehensive alternative to targeted SNP-based testing. This is particularly relevant as NGS data becomes more widely available and could provide the means for reducing Rh alloimmunization in children with SCA.

EXpanding Treatment for Existing Neurological Disease (EXTEND): An Open-Label Phase II Clinical Trial of Hydroxyurea Treatment in Sickle Cell Anemia.

BACKGROUND: Cerebral vasculopathy in sickle cell anemia (SCA) begins in childhood and features intracranial arterial stenosis with high risk of ischemic stroke. Stroke risk can be reduced by transcranial doppler (TCD) screening and chronic transfusion therapy; however, this approach is impractical in many developing countries. Accumulating evidence supports the use of hydroxyurea for the prevention and treatment of cerebrovascular disease in children with SCA. Recently we reported that hydroxyurea significantly reduced the conversion from conditional TCD velocities to abnormal velocities; whether hydroxyurea can be used for children with newly diagnosed severe cerebrovascular disease in place of starting transfusion therapy remains unknown. OBJECTIVE: The primary objective of the EXpanding Treatment for Existing Neurological Disease (EXTEND) trial is to investigate the effect of open label hydroxyurea on the maximum time-averaged mean velocity (TAMV) after 18 months of treatment compared to the pre-treatment value. Secondary objectives include the effects of hydroxyurea on serial TCD velocities, the incidence of neurological and non-neurological events, quality of life (QOL), body composition and metabolism, toxicity and treatment response, changes to brain magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA), genetic and serologic markers of disease severity, and cognitive and pulmonary function. METHODS: This prospective Phase II trial will enroll children with SCA in Jamaica, between the ages of 2 and 17 years, with either conditional (170-199 cm/sec) or abnormal (≥ 200 cm/sec) TCD velocities. Oral hydroxyurea will be administered daily and escalated to the maximum tolerated dose (MTD). Participants will be seen in the Sickle Cell Unit (SCU) in Kingston, Jamaica monthly until achieving MTD, and then every 3 months. TCD will be performed every 6 months. RESULTS: Currently, 43 participants have been enrolled out of a projected 50. There was one withdrawal due to immigration, with no permanent screen failures. Of the 43 enrolled, 37 participants have initiated study treatment. CONCLUSIONS: This trial investigates the effects of hydroxyurea treatment at MTD in children with conditional or abnormal TCD velocities before transfusion therapy and may represent an important advance towards establishing a suitable non-transfusion protocol for stroke prevention in children with SCA. The trial outcomes will have profound significance in developing countries where the disease burden is highest. CLINICALTRIAL: ClinicalTrials.gov NCT02556099; https://clinicaltrials.gov/ct2/show/NCT02556099 (Archived by WebCite at http://www.webcitation.org/6k1yMAa9G).

A Cost-Effectiveness Analysis of a Pilot Neonatal Screening Program for Sickle Cell Anemia in the Republic of Angola.

OBJECTIVE: To assess the cost-effectiveness of a pilot newborn screening (NBS) and treatment program for sickle cell anemia (SCA) in Luanda, Angola. STUDY DESIGN: In July 2011, a pilot NBS and treatment program was implemented in Luanda, Angola. Infants identified with SCA were enrolled in a specialized SCA clinic in which they received preventive care and sickle cell education. In this analysis, the World Health Organization (WHO) and generalized cost-effectiveness analysis methods were used to estimate gross intervention costs of the NBS and treatment program. To determine healthy life-years (HLYs) gained by screening and treatment, we assumed NBS reduced mortality to that of the Angolan population during the first 5 years based upon WHO and Global Burden of Diseases Study 2010 estimates, but provided no significant survival benefit for children who survive through age 5 years. A secondary sensitivity analysis with more conservative estimates of mortality benefits also was performed. The costs of downstream medical costs, including acute care, were not included. RESULTS: Based upon the costs of screening 36,453 infants and treating the 236 infants with SCA followed after NBS in the pilot project, NBS and treatment program is projected to result in the gain of 452-1105 HLYs, depending upon the discounting rate and survival assumptions used. The corresponding estimated cost per HLY gained is $1380-$3565, less than the gross domestic product per capita in Angola. CONCLUSIONS: These data demonstrate that NBS and treatment for SCA appear to be highly cost-effective across all scenarios for Angola by the WHO criteria.

Effects of chronic transfusions on abdominal sonographic abnormalities in children with sickle cell anemia.

OBJECTIVE: To assess the effects of chronic erythrocyte transfusions on prevalence of sonographic incidence of organ damage in children with sickle cell anemia (SCA). STUDY DESIGN: Children (N=148; mean age, 13.0 years) with SCA, receiving chronic transfusions (average, 7 years), underwent abdominal sonography at 25 institutions. After central imaging review, spleen, liver, and kidney measurements were compared with published normal values. Potential relations between ultrasound, clinical, and laboratory data were explored via analysis of variance, Student t test, and Cochran-Mantel-Haenzel tests of non-zero correlation. RESULTS: Average spleen length was similar to normal children, but over one-third had spleen volumes >300 mL, 15 had previous splenectomy for splenomegaly, and 24 had abnormal splenic echotexture. Two-thirds had hepatobiliary disease; 37 had prior cholecystectomy, 46 had gallstones, and 16 had gallbladder sludge. Gallbladder disease correlated with older age (P=.002), longer liver length (P<.001), longer duration of transfusions (P=.034), and higher total bilirubin (P<.001). Liver (P<.001) and renal lengths (P≤.005) were larger than published norms. CONCLUSIONS: In children with SCA, long-term transfusion therapy may not prevent development or progression of abdominal organ dysfunction.

Brown recluse spider (Loxosceles reclusa) envenomation leading to acute hemolytic anemia in six adolescents.

Loxosceles reclusa (brown recluse spider) bites often cause local envenomation reactions; however, acute hemolysis from systemic loxoscelism is rare. To highlight this important diagnostic consideration for unexplained hemolysis in areas endemic for brown recluse spiders, we report on 6 adolescents with acute hemolytic anemia from presumed L reclusa bites.

Renal function in infants with sickle cell anemia: baseline data from the BABY HUG trial.

OBJECTIVES: To examine the feasibility and accuracy of glomerular filtration rate (GFR) measurements in infants with sickle cell anemia (SCA). STUDY DESIGN: The NHLBI/NICHD-sponsored Phase III randomized double-blinded placebo-controlled trial (BABY HUG) tests the hypothesis that hydroxyurea can prevent chronic organ damage in SCA. GFR elevation is a coprimary endpoint, measured quantitatively by technetium 99m-labeled diethylenetriaminepentaacetic acid (DTPA) plasma clearance and estimated by the Schwartz equation with height and creatinine. RESULTS: Baseline DTPA GFR measurement was attempted in 191 infants; 176 of 184 completed studies (96%) were interpretable. Average age (mean +/- 1SD) was 13.7 +/- 2.6 months. Average DTPA GFR was 125.2 +/- 34.4 (range 40.2-300.9, normal 91.5 +/- 17.8 mL/min/1.73m(2)), while Schwartz estimates were higher at 184.4 +/- 55.5 mL/min/1.73m(2). DTPA GFR was correlated with Schwartz GFR (r(2) = 0.0658, P = .0012); also with age, weight, height, and kidney volume (all P < .002); but not with hemoglobin, HbF, white blood cell count, reticulocytes, medical events, or splenic function. CONCLUSIONS: Quantitative GFR measurement is feasible but variable among infants with SCA. Schwartz GFR estimates are not highly correlated with quantitative DTPA GFR values. Baseline GFR measurements suggest that renal dysfunction in SCA, evidenced by glomerular hyperfiltration, begins during infancy.

Prevention of secondary stroke and resolution of transfusional iron overload in children with sickle cell anemia using hydroxyurea and phlebotomy.

OBJECTIVE: Transfusions prevent secondary stroke in children with sickle cell anemia (SCA) but also cause iron overload. Alternatives for stroke prophylaxis with effective therapy to reduce iron burden are needed. STUDY DESIGN: For 35 children with SCA and stroke, transfusions were prospectively discontinued. Hydroxyurea was prescribed for stroke prophylaxis, and phlebotomy removed excess iron. Initial patients discontinued transfusions before hydroxyurea therapy, but later patients overlapped transfusions with hydroxyurea until tolerating full-dose therapy. RESULTS: Children received hydroxyurea for 42 +/- 30 months (range, 3-104 months). Hydroxyurea (26.7 +/- 4.8 mg/kg per day) led to mild neutropenia (3.9 +/- 2.3 x 10(9)/L) with significant increases in hemoglobin concentration, mean corpuscular volume, and fetal hemoglobin. Stroke recurrence rate was 5.7 events per 100 patient-years, but children receiving overlapping hydroxyurea therapy had only 3.6 events per 100 patient-years. For 26 children with >6 months of phlebotomy, 14,311 +/- 12,459 mL blood (315 +/- 214 mL/kg) was removed, with serum ferritin decreasing from a median of 2722 to 298 ng/mL. Among patients completing phlebotomy, liver biopsy documented normal histology and no excess iron deposition. CONCLUSIONS: For children with SCA and stroke, hydroxyurea effectively prevents secondary stroke and serial phlebotomy leads to complete resolution of transfusional iron overload.

Childhood autoimmune cytopenia secondary to unsuspected common variable immunodeficiency.

Immune thrombocytopenic purpura and autoimmune hemolytic anemia are typically idiopathic processes without underlying systemic illness. Four children with autoimmune cytopenia had low immunoglobulin levels that led to the diagnosis of common variable immunodeficiency. Routine screening of immunoglobulins is suggested for children with chronic or recurrent immune thrombocytopenic purpura and autoimmune hemolytic anemia.

Successful use of anti-CD20 (rituximab) in severe, life-threatening childhood immune thrombocytopenic purpura.

Thrombocytopenia with life-threatening hemorrhage in childhood immune thrombocytopenic purpura is rare, but effective therapeutic options are limited for the patient with bleeding. We report the efficacy of humanized anti-CD20 monoclonal antibody (rituximab, Rituxan) therapy for an infant with severe, refractory life-threatening immune thrombocytopenic purpura.

Effect of hydroxyurea on growth in children with sickle cell anemia: results of the HUG-KIDS Study.

OBJECTIVES: Although hydroxyurea is effective in treating adults with sickle-cell anemia (SCA), there is concern that it may adversely affect growth in children. We report the growth characteristics of patients in the Phase I-II pediatric hydroxyurea trial (HUG-KIDS) before and during treatment at the maximum tolerated dose for one year. STUDY DESIGN: Children and adolescents with SCA (n = 68), aged 5 to 16 years at baseline, reached the maximum tolerated dose and had serial height, weight, and Tanner stage measurements. Data from the Cooperative Study of Sickle Cell Disease (CSSCD) were used for comparison. Mixed-effects models were used to compare serial measurements as a function of age and group. RESULTS: In girls, there were no significant differences in height or weight among the pretreatment, on-treatment, and CSSCD groups. Compared with the CSSCD group, HUG-KIDS boys were heavier starting at age 9 years, and pretreatment HUG-KIDS boys were taller starting at age 7 years. The Tanner stage transitions took place at appropriate ages. CONCLUSIONS: Hydroxyurea treatment had no adverse effect on height or weight gain or pubertal development in school-aged children with SCA.