SNP Genotyping and LD Testing in ERMAP : Revealing Scianna Blood Group Diversity in NIH Blood Donors

Abstract Abstract 2322 Background: The Scianna blood group system has been implicated in cases of hemolytic disease of the fetus and newborn and the detection of antibodies to rare antigens in this system have impacted on transfusion management in some patients. Recently, it has been discovered that the Scianna blood group antigens are expressed by the erythrocyte membrane-associated protein (ERMAP), a 475 amino acid red cell adhesion protein consisting of 12 exons with the transcription region spanning exons 3–12. Rare variants in exons 4 and 12 have been reported in patients who have made antibodies to Scianna antigens or have a serological null phenotype for the Scianna system. ERMAP is a member of the butyrophilin-like family, featuring an extracellular immunoglobulin variable and intracellular B30.2 domains. Although one ERMAP variant is detected in one commercial molecular assay (Sc1/Sc2), most reported variants in this gene are rare, and therefore remain largely unrecognized during transfusion planning. ERMAP polymorphisms remain unreported on a large scale, contributing to the uncertainty concerning their clinical significance. To fill this void, we characterized seventeen single nucleotide polymorphisms (SNPs) in exons 3, 4, and 12 of ERMAP in 905 repeat blood donors. Methods: The DNA of consenting, repeat NIH blood donors were genotyped for seventeen variants in the ERMAP gene. DNA was isolated from whole blood using the Qiagen's MagAttract EZ1 kit. Following polymerase chain reaction amplification, the samples were genotyped by ligation detection reaction (LDR). LDR utilizes a thermostable ligase to generate single stranded DNA fragments of engineered length with allele-specific fluorescent labels, allowing for rapid, multiplexed genotyping. Ligated products were resolved by capillary electrophoresis (3730 DNA analyzer and GeneMapper software (Life Technologies)). Results: Eleven of the seventeen variants (G35S, R81Q, nt307Δ2, Q296Q, R332X, R392H, L399L, L409L, S442P, L452P, and L452L) were monomorphic in this cohort (N=905). Overall, the 54c>t and 76c>t transitions in exon 3 had minor allele frequencies (MAF) of 0.21 and 0.23, respectively, and appeared in all self-identified ethnic groups (except Native American donors (n=2)) with maxima observed in donors of self-identified Hispanic ethnicity (n=16; MAF=0.41 and 0.44, respectively). These SNPs showed significant linkage disequilibrium (r2=0.86 [95%CI 0.85–0.88]). African-American donors (n=57) had the highest frequency of variant 11c>t (MAF 0.07) and variant 755c>t (MAF 0.018), which was absent or extremely rare in other ethnic groups. The Caucasian donor population was the only group to display variations 788g>a and 1094g>a (MAF 0.003 and 0.0008 respectively). Conclusions: This is the largest sample of blood donors to be comprehensively genotyped for Scianna variants to date. We observed population-specific polymorphism of these rare variants according to the donor’s self-identified ethnicity, which is under further study. Determining the diversity in the Scianna blood group system may help explain otherwise unclear transfusion reactions, particularly if these variants impact on Scianna antigen surface density (especially the predicted leader sequence variants in exon 3) or other ERMAP functions (via variants in the intracellular domain encoded by exon 12). High throughput donor genotyping will allow evaluation of the clinical importance in alloimmunization for variants like the 11c>t, 54c>t, and 76c>t SNPs that lie in the predicted leader sequence and polymorphisms 755c>t, 788g>a, and 1094g>a that lie within the intracellular B30.2 domain of the ERMAP protein. Awareness of the frequencies of these variations can therefore be a clinically useful aid in the investigation of donors implicated in transfusion reactions. Disclosures: No relevant conflicts of interest to declare.