OA4-AM24-TU-15 - Targeted Quantitative NGS Uncovers 5 RH Alleles in Two Unrelated Patients, With DAU6 and Pseudogene in a Possible Tandem Arrangement

Significant homology within the RH locus has given rise to numerous hybrid alleles through gene conversion events, often resulting in altered phenotypes. We investigated the Rh genotype and phenotype of an 85-year-old Black female (S1) with C+ E‒ c+ e+ predicted by initial DNA testing, C‒ and variable e detected by serology, and possible anti-e-like reactivity in the plasma. Prompted by the genotyping results for S1, we investigated a sample from an unrelated individual (S2) with similar genetic features.

Study

Design/Methods: Genomic DNA was isolated from peripheral blood. Genotyping included: RHD Beadchip (Werfen); Sanger sequencing of RHD exons 7 and 8; targeted next-generation sequencing (NGS) of RH (Illumina) analyzed with custom-developed software. S1 analysis also included: RHCE BeadChip (Werfen); sequencing of RHCE exons 2, 4, 5, and RH*C exon 5; serology testing for D, C, c, E, e antigens with multiple reagents by tube method.

Results/Findings: Refer to Table 1 for serology results. S1 genotyping: RHD BeadChip detected RHD and RHD*Pseudogene (Psi); RHCE Beadchip detected RHCE*Ce and *ceHAR, failing to interpret c.48C/C. In contrast to RHD/RHCE BeadChips, Sanger sequencing was consistent with DAU6 and Psi in RHD; in RHCE exon 5 it detected c.659A and Psi-specific sequences, including c.654C and c.674T, all in heterozygosity. RH NGS detected RHD, DAU6, Psi in RHD, and CeN.06, novel ce(48C)-Psi(5)-D(9) in RHCE. S2 genotyping: Various results suggested RHD*DAU6 / Pseudogene(186T,410T,455C) (novel). RH NGS detected DAU6, Psi, DIIIa-ceVS.03(4-7)-D in RHD, and ceS, ce(48C)-D(9) in RHCE.



Conclusions: Accurate detection of complex genotypes can prevent the prediction of erroneous phenotypes. In S1, Sanger sequencing predicted a partial D phenotype, while RHCE Beadchip predicted C+ and e+ phenotypes, all discrepant with the serology. Targeted quantitative NGS detected three RHD alleles (including a common RHD), novel RHCE*ce(48C)-Psi(5)-D(9), rare RHCE*CeN.06, and correctly predicted D+, C−, and variable e phenotypes. Analogous to allele RHCE*ceAR, which encodes multiple ce-to-D amino acid changes in exon 5, hybrid RHCE*ce(48C)-Psi(5)-D(9) may encode a partial e antigen, consistent with the anti-e like reactivity detected in the plasma and variable e typing. Interestingly, a similar RHD/RHCE hybrid with RHD*Psi-c.807G-into-RHCE conversion was described in a Caucasian newborn (2022 Transfusion 62:E70). The patient’s health precludes autologous donation, and she has tolerated transfusion with C‒e‒ units matched also for K, Fy, and Jk. Remarkably, the genotype of S1 and S2 consisted of 5 RH alleles with RHD*DAU6 and RHD*Psi present in both, supporting a possible cis, in-tandem arrangement. Samples with more than four RH alleles such as the ones described here improve our understanding of the complexity at this locus, leading to more accurate genotype calls.