(P-IG-47) Three Previously-Undescribed Double Variant RHCE alleles

Background/Case Studies: New RHCE alleles are often identified by investigating discrepancies between serology and genotyping or even during routine patient genotyping. Resolving discrepancies is essential for accurate donor unit labeling or determining Rh antigen status in patients. We investigated a donor with a C antigen discrepancy between serology and genotyping (S1), a Sickle Cell Disease (SCD) patient sent for extended genotyping (S2), and a hemolytic anemia patient with warm anti-C autoantibody sent for RH genotyping (S3).

Study

Design/Methods: Serologic testing was performed by automated PK7400 and/or by standard tube method using the following anti-C and anti-c reagents: Immucor Gamma-clone (Werfen), BioClone (Ortho), ALBAclone (Alba), and Seraclone (Bio-Rad). An unlicensed human source anti-V/VS was used to type S2. Genomic DNA isolated from WBCs (QIAGEN) was analyzed using PreciseType HEA and/or RHCE BeadChip (Werfen). RH exons 1-10 and flanking intron regions were sequenced by targeted NGS and RHCE by Sanger sequencing. Allele-specific long-range PCR (AS-LR-PCR) was performed to phase variants.

Results/Findings: Table 1 summarizes the sample, ethnicity, serology and genotyping results.

S1 RBCs typed C– by PK7400 with discrepancy from predicted C+ by HEA. RBCs typed C– with all Anti-C reagents tested. RHCE BeadChip reported RHCE*CeCW/ce. However, CeCW is not predicted C–, prompting sequencing which revealed c.495C >A (p.Tyr165Ter). Sequencing of AS-LR-PCR phased c.495A to CeCW-specific c.122G.

S2 was RHCE*Ce/ceVS.01 by HEA; Ce prompted sequencing of exon 4 to rule out RHCE*CeRN. Changes for CeRN were not present, but rather c.545C >G (p.Thr182Ser) was identified. RHCE BeadChip and NGS confirmed changes for Ce/ceVS.01 with NGS also showing c.545C >G. The c.545G was phased to ceVS.01-specific c.733G by AS-LR-PCR. RBCs typed c+ (4+) with all reagents and V/VS+.

S3 RHCE BeadChip results reported RHCE*Ce/ce(1025T). The uncommon ce(1025T) prompted more testing. NGS confirmed BeadChip results and identified variant, c.73A >T (p.Ile25Phe), and phased it to RH*c. Linking c.73T to c.1025T could not be determined, though to date, c.1025T has only been seen on RHCE*ce. RBCs typed c+ (4+) with all anti-c.

Conclusions: We report three new alleles. RHCE*Ce(122G, 495A) encodes an early stop codon and therefore likely results in a null phenotype, though adsorption/elution studies couldn’t be done to confirm this. RHCE*ce(545G,733G) and ce(73T,1025T) are not associated with weak antigen expression, as shown by serology, but they harbor well-characterized variants (c.733G and c.1025T, respectively) known to lead to partial c and e antigens. The c.73T variant, when present on RHD, leads to a weak phenotype, but apparently not when on RHCE*ce, or when together with c.1025T, as evidenced by S3’s strong reactivity with anti-c.