ALBERT

Description: Introduction: In low-resource settings, red blood cell (RBC) transfusions for children with sickle cell anemia (SCA) may be cross-matched for only ABO and RhD antigens, so RBC alloimmunization can develop due to differences in Rh and other antigens between donors and recipients. In the Rh system, these differences can include absence of the antigen or the presence of partial or weak antigens, the latter more common with African ancestry. RBC alloimmunization in SCA has not been described in the Dominican Republic, and the genetic diversity of the RHD and RHCE loci have not been reported in this Hispanic-Afro-Caribbean population. Methods: A pediatric stroke prevention trial in the Dominican Republic (SACRED, NCT02769845) provided serum and dried blood spots from children with SCA, collected at enrollment and stored at -80C. Sera were screened for alloantibodies using a modified test-tube agglutination identification method with commercially available Panocell-20 (Immucor) RBC suspensions of known antigen phenotypes. Reaction patterns identified antigen specificity using standard blood bank methods. Patterns reactive with all cells tested were reported as pan-reactive and reactions without patterns were deemed low frequency antibodies. For all samples with an alloantibody, a matched SACRED control (age, sex, and transfusion history) was identified. Genomic DNA was analyzed by PCR and direct sequencing of each RHD and RHCE exon for single nucleotide polymorphisms (SNPs) that confer weak, partial, or variant phenotypes. Copy number variant PCR techniques with gene-specific primers were used to detect exonic substitutions in the RHD and RHCE gene loci that lead to negative or partial phenotypes. Results: A total of 272 SACRED participants were screened for RBC alloantibodies; 179 (66%) had at least one transfusion prior to enrollment, of whom 37 (21%) had a positive alloantibody screening result. Children with RBC alloantibodies (17 males, 20 females, average age 9.0 ± 2.4 years) had variable transfusion exposure with 23 (62%) having 〉10 prior transfusions, while 7 (19%) had ≤2 previous transfusions. Among the 37 positive samples, 18 sera had reactivity against Rh antigens including anti-E (12), anti-D (6, including 3 females), and anti-C specificity (5); 8 samples were consistent with a low frequency antibody and 3 were pan-reactive. Other alloantibodies had specificity to K (3, 1 female), Fya (4), Fyb (1), Lea (1), Leb (1), S (1), s (1), Jka (1), and U (1). More than one alloantibody was identified in 7 samples, of which 5 had two Rh antibodies. Genomic DNA analysis revealed numerous RHD variants, including several with one or more exonic SNPs that predict weak or partial RhD phenotypes, as well as predicted D-negative or partial phenotypes with hybrid RHD-CE-D genes or an insert pseudogene (RHDψ). Comparing the alloimmunized patients and controls, there was an equivalent frequency of RHD SNP variants (15/37 = 41%) in both cohorts, but more children with hybrid RHD-CE-D genes affecting multiple RHD exons (10 versus 4, p=0.077). All samples had the homozygous ccee genotype in the RHCE locus. There was a high frequency of RHCE SNP variants in both alloimmunized (31/37 = 84%) and control cohorts (32/37 = 86%), and only one control sample with possible RHD insertion in RHCE exon 5. All six children with anti-D alloantibodies had hybrid RHD-CE-D genes, typically typing as D-negative in tube testing. Among 17 children with anti-C or anti-E alloantibodies, 16 had at least one RHCE SNP, most frequently RHCE*ce.01, but no SNPs were associated with the development of Rh alloantibodies. Conclusions: RBC alloimmunization in children with SCA is common and often unrecognized in low-resource settings. In the Dominican Republic, 21% of children with SCA who entered SACRED with previous transfusions had RBC alloimmunization, including 10% with antibodies against Rh antigens. DNA analysis confirms extensive genetic complexity in both RHD and RHCE genes, with numerous SNPs that alter Rh antigen expression. Large RHD-CE-D insertions may also confer increased risks of anti-D alloimmunization. Lack of typing beyond serologic tube testing of ABO/RhD, and the presence of weak/partial RhD phenotypes in blood donors, may further increase the risk. Extending phenotyping for RhCE, providing RhD-negative blood when available, and performing enhanced screening for RhD-negative donors, will help reduce RBC alloimmunization. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Hydroxyurea is a potent therapeutic agent for sickle cell anemia (SCA), and treatment at maximum tolerated dose (MTD) is becoming the standard of care. Hydroxyurea exerts its disease-modifying effects primarily through induction of fetal hemoglobin (HbF), although the cellular and molecular mechanisms by which hydroxyurea increases HbF expression remain unclear. Children with SCA treated with hydroxyurea at MTD have substantial phenotypic variation, however, as some have higher HbF responses than others. We hypothesized that unknown quantitative trait loci modulate the pharmacological induction of HbF, so we performed a large genome wide association study (GWAS) of hydroxyurea-associated HbF responses for children with SCA treated prospectively with dose escalation to MTD. Methods: We analyzed genomic DNA from 831 children with SCA enrolled in pediatric research trials from the US (HUSTLE, SWiTCH, TWiTCH), the Caribbean (EXTEND, SACRED) and sub-Saharan Africa (REACH, NOHARM); all of these trials reported robust treatment responses with average HbF 〉20%. Study participants received hydroxyurea with dose escalation to MTD based on mild myelosuppression. Whole blood DNA was genotyped using the H3Africa SNP array (Illumina) with whole exome sequencing (WES) using NimbleGen VCRome 2.1 capture reagents and the Illumina HiSeq2500 platform. A transformed z-score for each study cohort gave a standardized measure of HbF induction relative to their steady-state level and their treatment HbF level at MTD. These standardized z-score HbF values were then used as a continuous variable for association testing using single-locus mixed model (EMMAX) adjusted for population stratification, using age, hydroxyurea dose at MTD, and sex as co-variates. We first performed an initial GWAS discovery using hydroxyurea response data from four distinct African populations (n=377). Single nucleotide variants (SNVs) with nominal significance (p

Description: Introduction: Elevated levels of fetal hemoglobin (HbF) are known to ameliorate both the morbidity and mortality of sickle cell anemia (SCA). Sustained post-natal HbF expression is heritable and regulated by multiple quantitative trait loci. Previous genomic studies have identified three major gene loci (BCL11A, HBS1L-MYB, and HBG2) that account for ~40% of HbF variation in SCA, but additional genetic modifiers remain to be discovered. We performed a genome wide association study (GWAS) using DNA collected from multiple cohorts of children with SCA, to identify novel genes and variants involved in HbF expression. Methods: We analyzed genomic DNA from 1009 children with SCA and pre-treatment steady-state HbF levels who enrolled in prospective research trials from the United States (HUSTLE, SWiTCH, TWiTCH), the Caribbean (SACRED) or sub-Saharan Africa (REACH, NOHARM). Whole blood DNA was first genotyped using the H3Africa SNP array (Illumina) that identifies over 2.2 million single nucleotide variants (SNVs) across the genome. Most samples also underwent whole exome sequencing (WES) using NimbleGen VCRome 2.1 capture reagents and the Illumina HiSeq2500 platform analysis, which identifies coding variants in all known exons. Square root transformed HbF values were the continuous variable for association testing using single-locus mixed model (EMMAX) adjusted for population stratification, with both age and sex as co-variates. The GWAS approach included 3 distinct steps. First, we performed two independent GWAS discovery steps using distinct African populations; these were designated Discovery I (N=211) and Discovery II (223). Second, only SNVs that were significant (p