ALBERT

Description: Abstract 2287 High throughput genomic testing for blood groups allows large scale antigen typing and assessment of donor pool compatibility with chronic transfusion dependent populations, particularly thalassemia or sickle cell disease (SCD) patients, in order to decrease RBC alloimmunization. Thus, it is crucial to determine if the quantity and antigen diversity of the donor pool meets the demands to sustain these patients on phenotype/genotype extended matched chronic transfusion protocols. We calculated the most common extended RBC predicted phenotypes for the 12 major clinically significant blood group antigens, D, CcEe, K, Jka/b, Fya/b, and Ss, in patients undergoing chronic transfusion for sickle cell disease (n=203) or thalassemia (n=98). The most common phenotypes in each diagnostic group were used to determine the prevalence of these phenotypes in a single day donor inventory (n=5,000) and stratified by ethnic group (70% Caucasian, 10% African-American, 13% Hispanic, 5% Asian, and 2% Other). All patient samples were tested for the presence of the GATA mutation which disrupts erythroid expression of Fy(b), and if present deemed not at risk for Fy(b) alloimmunization. The majority of patients with SCD and thalassemia are RhD positive (97% and 90% respectively), but differ in extended Rh phenotype, with Ro (Dce) prevalent in SCD pa tients (61%), and R1 (DCe) in thalassemia patients (79%). For patients with SCD, the most prevalent antigen-negative phenotypes were 17% C-E-K-,Fy(a-),Jk(b-), S-; 9% C-E-K-, Fy(a-),S-; 5% E-K-,Fy(a-),S-; and 4% C-K-,Fy(a-), Jk(b-), S-. In patients with thalassemia, no minor antigen profile exceeded 5% of individuals. The most prevalent antigen-negative profiles were 5% E-c-K-, Fy(b-),Jk(b-),S-, and 4% E-K-. Comparison of the most prevalent antigen-negative phenotype in patients with SCD with the donors revealed only 0.06% Caucasian (n=2), but 20% of the African-American donors (n=90) were antigen-negative matches. For the second most prevalent phenotype, 0.08% Caucasian (n=3) and 33% of African-American (n=167), 2% of Hispanic (n=13), and 5% other (n=5) were antigen-negative matches. For the third and fourth prevalent phenotypes, 47% of African-American (n=233) and 23% (n=115) respectively, were antigen-negative matches, while only 0.14% (n=5) and 0.06% (n=2) of Caucasians, but 5% (n=31) and 2% (n=13) of Hispanic donors were appropriate matches, respectively. For the thalassemia patients, antigen matches for the most common phenotype were found most often in Asian (13%) and donors identifying as “other” (6%). Matches were present in only 2% of the Caucasians, 2% of Hispanics, and 1.8% of African-American donors for thalassemia patients. These results confirmed the importance and impact of African-American donors for extended antigen-matching for patients with SCD. Less than 1% of our Caucasian donors could serve as extended matching for SCD. Nearly 20% of patients with SCD are negative for a common group of antigens, which allows future donor recruitment efforts focused on extended antigen profiles of the donor. Patients with thalassemia do not have a common antigen-negative profile, but extended matching for these patients can be improved by increase recruitment of Asian donors. High throughput genotyping enables typing of large numbers of donors, and potentially the majority of the donor inventory. Analysis of antigen-negative phenotypes in the donor pool with analysis of patient groups is important for inventory management, focused donor recruitment, and improved transfusion practice by avoiding alloimmunization. Disclosures: Stassinopoulos: Cerus: Employment, Equity Ownership, Patents & Royalties.

Description: Key Points RH genotyping of red cells may improve matching of patients and donors and reduce Rh alloimmunization. RH genotype matching may improve use of an African American blood donor inventory.

Description: Abstract 2040 The erythroid Rh family of proteins includes RhCE and RhD which carry the common Rh antigens, and the related Rh-associated glycoprotein, RhAG. RhAG is required for trafficking of the blood group proteins to the membrane and forms the core of a macro-complex in the membrane which includes glycophorin B, Band 3, CD47, and LW. The Rh proteins are structurally and functionally related to the Amt superfamily of NH3/NH4+ transport proteins, and RhAG and its nonerythroid paralogs, RhCG and RhBG, have been shown to mediate NH3/NH4+ transport. RhCG is responsible for part of renal collecting duct epithelial cell NH3/NH4+ secretion, and Rhcg-/- mice exhibit incomplete distal renal tubular acidosis due to impaired urinary NH4+ excretion. The Rhag-/- mouse is grossly normal, and the significance of RhAG-mediated NH3/NH4+ transport in human erythrocytes remains unclear. Over-hydrated hereditary stomatocytosis (OHSt) is a rare dominant disorder characterized by moderate hemolytic anemia, increased mean red cell volumes, stomatocytes and echinocytes, and increased red cell permeability to the monovalent cations, Na+ and K+. Six of the seven OHSt kindred studied by Bruce et al. (Blood. 2009;113:1350) displayed a heterozygous Phe65Ser mutation in RhAG. Expression studies of the mutant 65Ser-RhAG in Xenopus oocytes induced a monovalent cation flux compatible with the cation leak seen in RBCs. The increased Na+ and decreased K+ contents of mutant RhAG-expressing oocytes suggested that F65S is a gain-of-function mutation that opens a cation leak, likely within the RhAG polypeptide. In this study the ammonia transport properties of the OHSt mutant 65Ser-RhAG were investigated. Xenopus oocytes were injected with cRNA encoding wild-type RhAG, the OHSt mutant 65Ser-RhAG, and 65Val-RhAG, an engineered mutation with a smaller hydrophobic side chain at position 65. Wild-type and mutant RhAG polypeptides were well-expressed in the oocyte membrane as measured by quantitative immunoblotting. Uptake of the NH3/NH4+ substrate analog 14C-methylammonium (MA), was assayed in oocytes previously injected with water (control) or with cRNA. Expression of wild-type RhAG mediated MA uptake at rates 6-fold greater than that of water-injected controls. Uptake of MA by oocytes expressing 65Val-RhAG was equivalent to that of wild type RhAG. However, MA uptake by oocytes expressing OHSt mutant 65Ser-RhAG was greatly reduced to less than 20% that of oocytes expressing wild-type RHAG or 65Val-RhAG, and was only 1.5-fold greater than that of water-injected control oocytes. Co-expression with other, individual Rh complex members glycophorin B, RhD, RhCE, or Band 3 did not alter MA-mediated uptake by RhAG-expressing oocytes. Importantly, this study reveals that the RhAG mutation Phe65Ser found in patients with type 1 over-hydrated stomatocytosis is a loss of function mutation. Further study is required to define the relationship between loss of NH3/NH4+ transport and erythrocyte Na+ and K+ cation content. Disclosures: Westhoff: Immucor: Scientific Advisor.

Description: The erythrocyte Rh and Rh-associated (RhAG) proteins have distant sequence identity to a family of ammonium transporters found in yeast and bacteria. We previously showed that RhAG mediates movement of ammonium when expressed in yeast and in Xenopus oocytes. Importantly, these are the first mammalian proteins found to transport ammonium as a principal substrate. Elucidation of the mechanism and actual substrate(s) transported (protonated NH4+ or unprotonated NH3, or both) is important to understand their role in elimination of ammonium, proton recycling, and their impact on cellular pH and acid/base regulation. Functional characterization revealed that uptake was independent of the membrane potential and the Na+ gradient, but was dramatically stimulated by raising extracellular pH or lowering intracellular pH. This suggested that uptake was coupled to an outwardly directed H+ gradient and led us to hypothesize that RhAG might function by an H+-coupled, counter-transport mechanism. To further define the mechanism and actual substrate transported, RhAG-expressing oocytes were exposed to varying concentrations of NH4+ with constant NH3, and vice versa, by manipulation of the NH4Cl concentration and the pH of the buffer. A voltage-ramping protocol was used to evaluate changes in membrane conductance and reverse potential to measure membrane depolarization. Radioactive flux uptake of 14C-methylammonium, an analogue of ammonium, was used to measure transport. In the presence of substrate in the physiologic range (20 uM-500 uM), RhAG-mediated transport responded to the concentration of protonated NH4+ rather than the amount of unprotonated NH3 present. Currents in RhAG-expressing oocytes did not differ from water-injected controls. No significant changes in membrane conductance or membrane depolarization and reverse potential were observed. Taken together these data support a role for RhAG in the electronuetral transport of NH4+ by exchange with H+, and for erythrocytes in the maintenance of total blood ammonia levels. Sequestration of ammonium by erythrocytes would keep blood plasma levels low, preventing exposure of cells to toxic levels. Erythrocytes are ideally postioned to then transport ammonium to be exchanged in the liver and kidney, where other Rh-related proteins (RhBG and RhCG) are expressed.

Description: Introduction RBC alloimmunization is common in patients with sickle cell disease (SCD). Despite serological matching RBCs for major Rh antigens, Rh alloimmunization remains problematic. The Rh blood group is encoded by two genes RHD and RHCE, which exhibit extensive nucleotide polymorphism and chromosome structural changes, resulting in the formation of Rh variant antigens. Rh variants can result in loss of protein epitopes or expression of neo-epitopes, and are common in SCD patients. Hence SCD patients harboring Rh variants can be predisposed to Rh alloimmunization. Given the limitation of traditional serologic antigen typing for detection of Rh variants, molecular genotyping has become required. A DNA microarray-based platform, BioArray RHCE and RHD BeadChip (Immuncor) is available for RH genotyping. However, it detects the most common, but not all, variants. Whole exome sequence data have been used for prediction of Rh variants (Chou, et. al, Blood Adv., 2017), offer some advantages, including detection of rare variants, structural rearrangements and copy number variation. However, whole genome sequence (WGS) analysis of RHD/RHCE is challenging due to difficulties in mapping next generation sequencing (NGS) reads to this duplicated gene family. We developed a computational algorithm to identify RH variants using WGS data. Methods The pipeline included three major components, RH allele database construction, RH variant calling, and classification of Rh blood group according the identified variants. The RH allele database was built based on NCBI Blood Group Antigen Gene Mutation (BGMUT) and International Society of Blood Transfusion (ISBT) database. Since the alleles in the BGMUT and ISBT databases were specified according to conventional RH genes (RHD, L08429; RHCE, DQ322275) that are different from those on reference human genome, we first called the variations based on the reference human genome. The positions of the identified variations were subsequently corrected to match with the BGMUT and ISBT annotation system. Next, the NGS reads with low base quality and/or mapping quality were discarded during the variation calling step. Synonymous and non-synonymous amino acid changes were characterized for each polymorphism. Haplotypes were constructed for the segments with NGS read support. Gene sequencing coverage was calculated to determine gene deletions or amplifications. Lastly, we implemented an algorithm to predict RH genotypes based on a selection of candidate alleles by read-mapping profile which considers both sequence variations and sequence consistency followed by a likelihood-based ranking of all pairwise combinations of the selected alleles. The allele combination with the highest likelihood is considered the most likely pair of alleles at a given locus. Patient specimens used in this study were from participants of the Sickle Cell Clinical Research and Intervention Program (SCCRIP, Hankins et al. Pediatr Blood Cancer. 2018). Results We validated our method in a cohort of 58 SCD patients whose RH genotypes had been determined by BioArray RhCE and RhD BeadChip and supplementary molecular tests that identify the most common variants among individuals of African descent. In this validation cohort including a total of 11 RHD and 13 RHCE alleles, our approach achieved a concordance rate of 85.85% (91 of 106 alleles) for RHD and 83.02% (88 of 106 alleles) for RHCE genotyping. WGS was highly sensitive in distinguishing homozygosity from heterozygosity of genes. By comparing the numbers of NGS reads on RH regions and whole genome average coverage, heterozygous deletion can be determined. Since WGS provides comprehensive genotyping, our analysis identified single nucleotide polymorphisms that were not identified by the BeadChip and supplemental molecular testing. The final source of discordance was likely due to the short read length of NGS such that haplotype phases cannot be correctly predicted if the variations are separated by thousands of base pairs, for which long read DNA sequencing or RNA/cDNA sequencing are required. Evaluation of the identified discrepancies is ongoing. Conclusions We developed and validated a diagnostic method for RH genotyping that leveraged the accuracy and flexibility of RH genotyping based on WGS data. With further optimization of our method, this may be useful for RBC genotype matching sickle cell patients to blood donors in the future. Disclosures Hankins: Novartis: Research Funding; Global Blood Therapeutics: Research Funding; NCQA: Consultancy; bluebird bio: Consultancy.

Description: Abstract 3239 Red blood cell (RBC) transfusion is a key treatment for patients with sickle cell disease (SCD), but remains complicated by the high incidence of RBC immunization. Despite provision of phenotypic Rh D, C, and E antigen-matched donor RBCs, patients continue to develop Rh antibodies. In many cases, these antibodies are considered autoantibodies with specificities for D, C, and e antigens because the patient's own RBCs type serologically positive for the corresponding antigen. Recent evidence is emerging that Rh alloimmunization within populations of African origin is complicated by the genetic diversity of this locus. Individuals of African ancestry often carry RH alleles that differ from those defined as conventional alleles that are common in Europeans and other ethnic groups. These “variant” alleles encode Rh proteins, often with multiple amino acid changes, that either lack common epitopes or produce novel immunogenic epitopes. The clinical significance of Rh alloimmunization in patients with SCD with variant RH genes is largely unknown. In the present study, we performed RH genotyping in 212 patients with SCD to determine prevalence of RH variants, the association with Rh alloimmunization, and the clinical significance measured by changes in hematologic parameters at time of antibody detection. RH genotyping was performed by polymerase chain reaction (PCR) amplification using RHD-specific and RHCE-specific primers designed in the flanking intronic regions and analyzed by direct sequencing of exons, and/or a combination of multiple PCR-restriction fragment length polymorphism (RFLP) assays and by RHD and RHCE BeadChip arrays. We identified variant RH alleles in 88.7% (188/212) of patients with SCD. Twelve different RHD and 13 RHCE alleles encoding variant Rh D, C, and e antigens were represented in this cohort of patients. In 172 patients with 〉1 RBC transfusion (median 125 units), 55 antibodies were identified with Rh specificity despite antigen-positive status (28 anti-D, 16 anti-e, 9 anti-C, and 2 anti-E). RH genotypes revealed 47.3% of these antibodies developed in patients who lacked the corresponding conventional RH allele, and would be considered Rh alloantibodies. In 43.6%, RH genotypes predicted expression of the conventional antigen to which the antibody was directed, suggesting these were potentially autoantibodies. However, these patients had at least one other RH allele that was altered. This may suggest that the presence of one variant protein potentially changes the Rh complex in the membrane, and carrying at least one conventional RH allele is not necessarily protective against production of Rh specific antibodies. In the remaining 9.1%, we detected no variant RHD or RHCE alleles, and complete gene sequencing is in progress to confirm the absence of novel mutations. Importantly, to determine clinical significance, we evaluated whether Rh antibodies in patients carrying variant alleles caused decreased transfused RBC survival by comparing the patient's hematologic parameters at the time of antibody detection with the baseline pre-transfusion parameters. In all but 4 cases, Rh antibodies in antigen-positive patients occurred in chronically transfused patients. Therefore, baseline values were determined from the average pre-transfusion hemoglobin, hematocrit and % hemoglobin S level for the 6–12 months preceding antibody detection. Compromised transfused RBC survival was determined by a lower hemoglobin/hematocrit or higher % hemoglobin S level at time of antibody formation compared to the patient's baseline. Forty percent of antibodies were associated with delayed hemolytic transfusion reactions or decreased transfused RBC survival. Our data suggest that the high prevalence of variant RH alleles in patients with SCD is associated with clinically significant immunization. Discrimination of allo- versus auto- antibodies in this chronically transfused patient population presents a significant technical challenge and suggests a role for RH genotyping in the clinical evaluation of Rh antibodies and to improve RBC matching. Importantly, in this study Rh antibodies in patients with variant RH often compromised transfused RBC survival and, therefore, were clinically significant and may be targets for prevention strategies analogous to standard phenotype matching for C, E, and K. Disclosures: No relevant conflicts of interest to declare.