Thad A. Howard

Burden of sickle cell trait and disease in the Uganda Sickle Surveillance Study (US3): a cross-sectional study

BACKGROUND Sickle cell disease contributes substantially to mortality in children younger than 5 years in sub-Saharan Africa. In Uganda, 20,000 babies per year are thought to be born with sickle cell disease, but accurate data are not available. We did the cross-sectional Uganda Sickle Surveillance Study to assess the burden of disease. METHODS The primary objective of the study was to calculate prevalence of sickle cell trait and disease. We obtained punch samples from dried blood spots routinely collected from HIV-exposed infants in ten regions and 112 districts across Uganda for the national Early Infant Diagnosis programme. Haemoglobin electrophoresis by isoelectric focusing was done on all samples to identify those from babies with sickle trait or disease. FINDINGS Between February, 2014, and March, 2015, 99,243 dried blood spots were analysed and results were available for 97,631. The overall number of children with sickle cell trait was 12,979 (13·3%) and with disease was 716 (0·7%). Sickle cell numbers ranged from 631 (4·6%) for trait and 23 (0·2%) for disease of 13,649 in the South Western region to 1306 (19·8%) for trait and 96 (1·5%) for disease of 6581 in the East Central region. Sickle cell trait was seen in all districts. The lowest prevalence was less than 3·0% in two districts. Eight districts had prevalence greater than 20·0%, with the highest being 23·9%. Sickle cell disease was less common in children older than 12 months or who were HIV positive, which is consistent with comorbidity and early mortality. INTERPRETATION Prevalence of sickle cell trait and disease were high in Uganda, with notable variation between regions and districts. The data will help to inform national strategies for sickle cell disease, including neonatal screening. FUNDING Cincinnati Childrens Research Foundation.

Whole Exome Sequencing Identifies Novel Genes for Fetal Hemoglobin Response to Hydroxyurea in Children with Sickle Cell Anemia

Hydroxyurea has proven efficacy in children and adults with sickle cell anemia (SCA), but with considerable inter-individual variability in the amount of fetal hemoglobin (HbF) produced. Sibling and twin studies indicate that some of that drug response variation is heritable. To test the hypothesis that genetic modifiers influence pharmacological induction of HbF, we investigated phenotype-genotype associations using whole exome sequencing of children with SCA treated prospectively with hydroxyurea to maximum tolerated dose (MTD). We analyzed 171 unrelated patients enrolled in two prospective clinical trials, all treated with dose escalation to MTD. We examined two MTD drug response phenotypes: HbF (final %HbF minus baseline %HbF), and final %HbF. Analyzing individual genetic variants, we identified multiple low frequency and common variants associated with HbF induction by hydroxyurea. A validation cohort of 130 pediatric sickle cell patients treated to MTD with hydroxyurea was genotyped for 13 non-synonymous variants with the strongest association with HbF response to hydroxyurea in the discovery cohort. A coding variant in Spalt-like transcription factor, or SALL2, was associated with higher final HbF in this second independent replication sample and SALL2 represents an outstanding novel candidate gene for further investigation. These findings may help focus future functional studies and provide new insights into the pharmacological HbF upregulation by hydroxyurea in patients with SCA.

Characteristics of a rapid, point‐of‐care lateral flow immunoassay for the diagnosis of sickle cell disease

Sickle cell disease (SCD) is a common and life‐threatening hematological disorder, affecting approximately 400,000 newborns annually worldwide. Most SCD births occur in low‐resource countries, particularly in sub‐Saharan Africa, where limited access to accurate diagnostics results in early mortality. We evaluated a prototype immunoassay as a novel, rapid, and low‐cost point‐of‐care (POC) diagnostic device (Sickle SCAN™) designed to identify HbA, HbS, and HbC. A total of 139 blood samples were scored by three masked observers and compared to results using capillary zone electrophoresis. The sensitivity (98.3–100%) and specificity (92.5–100%) to detect the presence of HbA, HbS, and HbS were excellent. The test demonstrated 98.4% sensitivity and 98.6% specificity for the diagnosis of HbSS disease and 100% sensitivity and specificity for the diagnosis of HbSC disease. Most variant hemoglobins, including samples with high concentrations of HbF, did not interfere with the ability to detect HbS or HbC. Additionally, HbS and HbC were accurately detected at concentrations as low as 1–2%. Dried blood spot samples yielded clear positive bands, without loss of sensitivity or specificity, and devices stored at 37°C gave reliable results. These analyses indicate that the Sickle SCAN POC device is simple, rapid, and robust with high sensitivity and specificity for the detection of HbA, HbS, and HbC. The ability to obtain rapid and accurate results with both liquid blood and dried blood spots, including those with newborn high‐HbF phenotypes, suggests that this POC device is suitable for large‐scale screening and potentially for accurate diagnosis of SCD in limited resource settings. Am. J. Hematol. 91:205–210, 2016.

Genetic modifiers of white blood cell count, albuminuria and glomerular filtration rate in children with sickle cell anemia

Discovery and validation of genetic variants that influence disease severity in children with sickle cell anemia (SCA) could lead to early identification of high-risk patients, better screening strategies, and intervention with targeted and preventive therapy. We hypothesized that newly identified genetic risk factors for the general African American population could also impact laboratory biomarkers known to contribute to the clinical disease expression of SCA, including variants influencing the white blood cell count and the development of albuminuria and abnormal glomerular filtration rate. We first investigated candidate genetic polymorphisms in well-characterized SCA pediatric cohorts from three prospective NHLBI-supported clinical trials: HUSTLE, SWiTCH, and TWiTCH. We also performed whole exome sequencing to identify novel genetic variants, using both a discovery and a validation cohort. Among candidate genes, DARC rs2814778 polymorphism regulating Duffy antigen expression had a clear influence with significantly increased WBC and neutrophil counts, but did not affect the maximum tolerated dose of hydroxyurea therapy. The APOL1 G1 polymorphism, an identified risk factor for non-diabetic renal disease, was associated with albuminuria. Whole exome sequencing discovered several novel variants that maintained significance in the validation cohorts, including ZFHX4 polymorphisms affecting both the leukocyte and neutrophil counts, as well as AGGF1, CYP4B1, CUBN, TOR2A, PKD1L2, and CD163 variants affecting the glomerular filtration rate. The identification of robust, reliable, and reproducible genetic markers for disease severity in SCA remains elusive, but new genetic variants provide avenues for further validation and investigation.

Realizing effectiveness across continents with hydroxyurea: Enrollment and baseline characteristics of the multicenter REACH study in Sub-Saharan Africa.

Despite its well‐described safety and efficacy in the treatment of sickle cell anemia (SCA) in high‐income settings, hydroxyurea remains largely unavailable in sub‐Saharan Africa, where more than 75% of annual SCA births occur and many comorbidities exist. Realizing Effectiveness Across Continents with Hydroxyurea (REACH, ClinicalTrials.gov NCT01966731) is a prospective, Phase I/II open‐label trial of hydroxyurea designed to evaluate the feasibility, safety, and benefits of hydroxyurea treatment for children with SCA in four sub‐Saharan African countries. Following comprehensive training of local research teams, REACH was approved by local Ethics Committees and achieved full enrollment ahead of projections with 635 participants enrolled over a 30‐month period, despite half of families living >12 km from their clinical site. At enrollment, study participants (age 5.4 ± 2.4 years) had substantial morbidity, including a history of vaso‐occlusive pain (98%), transfusion (68%), malaria (85%), and stroke (6%). Significant differences in laboratory characteristics were noted across sites, with lower hemoglobin concentrations (P < .01) in Angola (7.2 ± 1.0 g/dL) and the DRC (7.0 ± 0.9 g/dL) compared to Kenya (7.4 ± 1.1 g/dL) and Uganda (7.5 ± 1.1 g/dL). Analysis of known genetic modifiers of SCA demonstrated a high frequency of α‐thalassemia (58.4% with at least a single α‐globin gene deletion) and G6PD deficiency (19.7% of males and 2.4% of females) across sites. The CAR β‐globin haplotype was present in 99% of participants. The full enrollment to REACH confirms the feasibility of conducting high‐quality SCA research in Africa; this study will provide vital information to guide safe and effective dosing of hydroxyurea for children with SCA living in Africa.

Hemoglobin variants identified in the Uganda Sickle Surveillance Study

The Uganda Sickle Surveillance Study analyzed dried blood spots that were collected from almost 100 000 infants and young children from all 10 regions and 112 districts in the Republic of Uganda, with the primary objective of determining the prevalence of sickle cell trait and disease. An overall prevalence of 13.3% sickle cell trait and 0.7% sickle cell disease was recently reported. The isoelectric focusing electrophoresis technique coincidentally revealed numerous hemoglobin (Hb) variants (defined as an electrophoresis band that was not Hb A, Hb F, Hb S, or Hb C) with an overall country-wide prevalence of 0.5%, but with considerable geographic variability, being highest in the northwest regions and districts. To elucidate these Hb variants, the original isoelectric focusing (IEF) gels were reviewed to identify and locate the variant samples; corresponding dried blood spots were retrieved for further testing. Subsequent DNA-based investigation of 5 predominant isoelectric focusing patterns identified 2 α-globin variants (Hb Stanleyville II, Asn78Lys; Hb G-Pest, Asp74Asn), 1 β-globin variant (Hb O-Arab, Glu121Lys), and 2 fusion globin variants (Hb P-Nilotic, β31-δ50; Hb Kenya, Aγ81Leu-β86Ala). Compound heterozygotes containing an Hb variant plus Hb S were also identified, including both Hb S/O-Arab and HbS/Kenya. Regional differences in the types and prevalence of these hemoglobin variants likely reflect tribal ancestries and migration patterns. Algorithms are proposed to characterize these Hb variants, which will be helpful for emerging neonatal hemoglobinopathy screening programs that are under way in sub-Saharan Africa.