CD59 gene: 143 haplotypes of 22,718 nucleotides length by computational phasing in 113 individuals from different ethnicities.

BACKGROUND: CD59 deficiency due to rare germline variants in the CD59 gene causes disabilities, ischemic strokes, neuropathy, and hemolysis. CD59 deficiency due to common somatic variants in the PIG-A gene in hematopoietic stem cells causes paroxysmal nocturnal hemoglobinuria. The ISBT database lists one nonsense and three missense germline variants that are associated with the CD59-null phenotype. To analyze the genetic diversity of the CD59 gene, we determined long-range CD59 haplotypes among individuals from different ethnicities. METHODS: We determined a 22.7 kb genomic fragment of the CD59 gene in 113 individuals using next-generation sequencing (NGS), which covered the whole NM_203330.2 mRNA transcript of 7796 base pairs. Samples came from an FDA reference repository and our Ethiopia study cohorts. The raw genotype data were computationally phased into individual haplotype sequences. RESULTS: Nucleotide sequencing of the CD59 gene of 226 chromosomes identified 216 positions with single nucleotide variants. Only three haplotypes were observed in homozygous form, which allowed us to assign them unambiguously as experimentally verified CD59 haplotypes. They were also the most frequent haplotypes among both cohorts. An additional 140 haplotypes were imputed computationally. DISCUSSION: We provided a large set of haplotypes and proposed three verified long-range CD59 reference sequences, based on a population approach, using a generalizable rationale for our choice. Correct long-range haplotypes are useful as template sequences for allele calling in high-throughput NGS and precision medicine approaches, thus enhancing the reliability of clinical diagnostics. Long-range haplotypes can also be used to evaluate the influence of genetic variation on the risk of transfusion reactions or diseases.

RH genotypes and red cell alloimmunization rates in chronically transfused patients with sickle cell disease: A multisite study in the USA.

BACKGROUND: Red cell alloimmunization remains a challenge for individuals with sickle cell disease (SCD) and contributes to increased risk of hemolytic transfusion reactions and associated comorbidities. Despite prophylactic serological matching for ABO, Rh, and K, red cell alloimmunization persists, in part, due to a high frequency of variant RH alleles in patients with SCD and Black blood donors. STUDY DESIGN AND METHODS: We compared RH genotypes and rates of alloimmunization in 342 pediatric and young adult patients with SCD on chronic transfusion therapy exposed to >90,000 red cell units at five sites across the USA. Genotyping was performed with RHD and RHCE BeadChip arrays and targeted assays. RESULTS: Prevalence of overall and Rh-specific alloimmunization varied among institutions, ranging from 5% to 41% (p = .0035) and 5%-33% (p = .0002), respectively. RH genotyping demonstrated that 33% RHD and 57% RHCE alleles were variant in this cohort. Patients with RHCE alleles encoding partial e antigens had higher rates of anti-e identified than those encoding at least one conventional e antigen (p = .0007). There was no difference in anti-D, anti-C, or anti-E formation among patients with predicted partial or altered antigen expression compared to those with conventional antigens, suggesting that variant Rh on donor cells may also stimulate alloimmunization to these antigens. DISCUSSION: These results highlight variability in alloimmunization rates and suggest that a molecular approach to Rh antigen matching may be necessary for optimal prevention of alloimmunization given the high prevalence of variant RH alleles among both patients and Black donors.

RHD-negative red cells may be avoided for patients with ambiguous serologic typing for the RHD antigen.

BACKGROUND AND OBJECTIVES: Serologic typing with monoclonal anti-D is mandatory for RHD antigen determination before transfusion, but due to aberrant (weak or partial) variants of RHD, results may be ambiguous and molecular RHD-typing is required. Before that, RHD-negative (RHD -) red blood cells concentrates (RBCs) shall be transfused to avoid anti-D formation, which probably leads to wastage of RHD - RBCs. STUDY DESIGN AND METHODS: All patients with ambiguous results in serologic RHD-typing and molecular RHD-typing were assessed retrospectively. The proportions of patients at risk for anti-D formation and the proportion of RHD - RBCs transfused unnecessarily were evaluated for the following transfusion strategies: (1) RHD-positive (RHD + )RBCs for all patients, (2) RHD + RBCs for patients with at least 2+ reaction with anti-D, (3) RHD + RBCs for patients with C and/or E in their RHCE-phenotype, (4) RHD + RBCs for patients with C and/or E and at least 2+ reaction, and (5) RHD - RBCs for all patients. RESULTS: A total of 112 patients were included. Most had weak D type 1-3 and a minority had other, rare RHD variants. The risk of anti-D formation was 4.5%, 2.9%, 1.8%, 1.0%, and 0% for strategies 1-5, respectively. The proportion of RHD - RBCs transfused unnecessarily was 0%, 49.5%, 0.9%, 50.5%, and 95.5%. CONCLUSION: Transfusing patients with a C and/or E in their RHCE-phenotype with RHD + RBCs resulted in a very low risk of immunization while avoiding wastage of RHD - RBCs. Therefore, this strategy should be used for some patients with ambiguous results in serologic RHD-typing and pending results of molecular RHD-typing.

Molecular and structural characterization of a novel high-prevalence antigen of the Augustine blood group system.

BACKGROUND: An antibody directed against a high-prevalence red blood cell (RBC) antigen was detected in a 67-year-old female patient of North African ancestry with a history of a single pregnancy and blood transfusion. So far, the specificity of the proband's alloantibody remained unknown in our immunohematology reference laboratory. STUDY DESIGN AND METHODS: Whole-exome sequencing (WES) was performed on the proband's DNA. The reactivity to the SLC29A1-encoded ENT1 adenosine transporter was investigated by flow cytometry analyses of ENT1-expressing HEK293 cells, and RBCs from Augustine-typed individuals. Erythrocyte protein expression level, nucleoside-binding capacity, and molecular structure of the proband's ENT1 variant were further explored by western blot, flow cytometry, and molecular dynamics calculations, respectively. RESULTS: A missense variant was identified in the SLC29A1 gene, which encodes the Augustine blood group system. It arises from homozygosity for a rare c.242A > G missense mutation that results in a nonsynonymous p.Asn81Ser substitution within the large extracellular loop of ENT1. Flow cytometry analyses demonstrated that the proband's antibody was reactive against HEK-293 cells transfected with control but not proband's SLC29A1 cDNA. Consistent with this finding, proband's antibody was found to be reactive with At(a-) (AUG:-2), but not AUG:-1 (null phenotype) RBCs. Data from structural analysis further supported that the proband's p.Asn81Ser variation does not alter ENT1 binding of its specific inhibitor NBMPR. CONCLUSION: Our study provides evidence for a novel high-prevalence antigen, AUG4 (also called ATAM after the proband's name) in the Augustine blood group system, encoded by the rare SLC29A1 variant allele AUG*04 (c.242A > G, p.Asn81Ser).