(P-IG-14) Blood Donor with C Typing Discrepancy Due to RHCE Allele with Novel In/del Variant

Background/Case Studies: Use of an RBC genotyping panel for screening blood donors is an efficient and cost-effective strategy to characterize their red cell phenotype. The predicted phenotypes have been found to be more accurate than serologic testing. However, low- and medium-resolution genotyping cannot rule out rare or novel variants that can impact phenotype. Discrepancies that arise between genotype-predicted phenotype and serologic phenotype often lead to the identification of rare or novel alleles. We describe a case where molecular testing predicted a blood donor to type C+ while RBCs typed C- during routine serologic typing using manual tube testing.

Study

Design/Methods: Blood specimens from blood donors were tested using HemoID DQS (DQS, Agena Bioscience) as part of routine screening program. Discrepancies were identified by automated concordance check between molecular and serologic results. DNA was tested using RHCE BeadChip (Werfen) with BASIS4G software. RHCE exons were amplified, and Sanger sequencing performed (Azenta) and sequences aligned to reference (Sequencher). ISBT allele table and public databases (dbSNP) were reviewed. Mutalyzer was used to determine the impact on protein translation.

Results/Findings: Medium-resolution genotyping using RHCE BeadChip predicted the phenotype to be C+ c+ E- e+ V- VS-, concordant with DQS. High-resolution sequencing confirmed the presence of the sequences found in RHCE*Ce allele. In addition, the donor was found to be heterozygous for a single base deletion in exon 6 at c.855. The variant was not found in the public databases. The coding and protein coordinates predict that the protein has a shift in reading frame and truncates two amino acids after the deletion: NC_000001.11(NP_065231.4):p.(His286ThrfsTer2) which is predicted to be in the fifth extracellular loop. This drastic alteration in the polypeptide structure likely impacts protein folding, membrane insertion and stability, resulting in a presumed null phenotype. Since the RHCE*Ce allele was coinherited with an RHCE*ce allele, the loss of e antigen expression cannot be assessed.

Conclusions: Many blood centers are using RBC genotyping to predict extended phenotypes. Discrepancies between predicted phenotype and serologic phenotype are important to investigate and resolve. In this case, a very rare in/del was discovered that is predicted to result in a truncated protein and is associated with loss of antigen expression.