Genetic Basis of Erythrocyte Alloimmunization Among Children with Sickle Cell Anemia in the Dominican Republic

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), Fy^a (4), Fy^b (1), Le^a (1), Le^b (1), S (1), s (1), Jk^a (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 (RHD ψ). 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.