American Red Cross Biomedical Services Philadelphia, Pennsylvania, United States
Background/Case Studies: Two blood donors (D1 and D2) were tested using HemoID DQS, a low-resolution red cell genotyping panel (DQS, Agena Bioscience) to obtain an extended red cell phenotype. D1 typed E- by tube testing on previous donations but DQS predicted C- c+ E+ e+. D2 typed c- E- on the Ortho Vision analyzer (Quidel Ortho) and manual tube testing but DQS predicted C+ c+ E+ e+. Additional molecular testing was performed to resolve these discrepancies.
Study
Design/Methods: Blood specimens from the two blood donors were extracted and genomic DNA tested using RHCE BeadChip (Werfen) and Sanger sequencing of RHCE exons 1-8. Resulting sequences were aligned to the reference sequence using Sequencher software (GeneCodes). PolyPhen-2 (Adzhubei Nat Methods 2010), a bioinformatics tool that uses structural and comparative evolutionary considerations to predict the possible impact of amino acid variants, was used to evaluate the variant identified in D2.
Results/Findings: Medium-resolution RHCE genotyping of D1 predicted the donor to carry RHCE*ce and RHCE*cE alleles and express c, e and E antigens concordant with DQS. High-resolution Sanger sequencing identified a single nucleotide variation (SNV) in RHCE exon 6 at c.933C >A which is predicted to result in premature translational termination at amino acid position p.311. Medium-resolution RHCE genotyping of D2 predicted the donor to carry RHCE*cE and RHCE*Ce alleles and express c, C, e and E antigens concordant with DQS.Sanger sequencing identified an SNV in RHCE exon 2 at c.281T >C that replaces leucine withproline at amino acid position p.94. Protein modeling predicted the impact of p.94Pro to be damaging with a score of 0.999. Conclusions: The two donors carry previously unreported SNVs that likely have nullifying effects on antigen expression. Neither of these SNVs have frequencies in dbSNP (NCBI). D1 carries a nonsense SNV c.933C >A, likely carried on the RHCE*cE allele, which is predicted to result in a truncated protein. D2 carries a non-synonymous SNV c.281T >C, likely carried on the RHCE*cE allele. Modeling of the protein encoded by this allele predicts the RhcE with proline at p.94 to be deleterious with high probability. Both the RHCE*cE933A (D1) and RHCE*cE281T (D2) alleles are very rare.
