New York Blood Center Enterprises, Immunohematology and Genomics Laboratory, New York, United States
Background/Case Studies: The Rh blood group system challenges the field of transfusion medicine by being extremely immunogenic and highly polymorphic. Variant alleles may encode weak, partial or null phenotypes and can be associated with alloantibody production. We identified three new RHD variant alleles in patient samples referred for genotyping due to weak or variable D typing results, two of them also with anti-D in plasma.
Study
Design/Methods: Serology was performed by the tube method with multiple Anti-D reagents (Table 1). Genomic DNA was isolated from WBCs (QIAGEN). Genotyping included hybrid Rhesus box assay to determine RHD zygosity, RHD BeadChip (Werfen), an NGS assay targeting both RH loci, and Sanger sequencing to confirm new variants found by NGS. RHD long-range allele-specific PCR (LR-AS-PCR) was done to link variant alleles when needed.
Results/Findings: Table 1 summarizes serology and genotyping results of the three samples. In all cases, RHD BeadChip did not detect variants to explain the altered D phenotypes. S1 was from a 76yo white Jewish male and Anti-D was detected in patient’s plasma . RBCs were weakly reactive at IS and strongly reactive at IAT. The hybrid Rhesus box assay revealed one RHD gene, and NGS linked a new variant c.483C >A (p.Phe161Leu) to it. S2 was from a 24yo female with weakly detectable anti-D in plasma. RBCs displayed variable reactivity with Anti-D reagents, which did not intensify significantly at IAT. NGS revealed RHD and RHD*Pseudogene alleles, along with new variant c.1028A >G (p.Tyr343Cys). This variant was linked to the RHD allele via LR-AS-PCR. S3 was from a 3yo male with sickle cell disease (SCD) whose RBCs exhibited a wide range of agglutination strengths at IS (mi to 3+) with different Anti-D reagents. NGS revealed RHD*Weak D type 4.0, common DAU variant c.1136C >T and rare c.993C >A (p.Asn331Lys). These two latter variants were linked using LR-AS-PCR.
Conclusions: We report three new RHD variant alleles: RHD*483A, RHD*1028G and RHD*993A,1136T in samples demonstrating a weak or variable D phenotype. Of note, Gamma-clone Anti-D reacted the strongest at IS with all samples and use of this reagent alone for S3 testing could have precluded the discovery of RHD*993A,1136T. Interestingly, c.993C >A is reported in a similar allele with variable D typing (RHD*165T,993A) and also mirrors the c.993C >G found in weak D type 90; all encode p.Asn331Lys. RHD*483A and RHD*1028G likely result in a partial D phenotype, given the variable reactivity across reagents, and the anti-D detected in patient plasma. RHD*1028G also occurs in a woman of child-bearing age and may affect RhIG administration in event of pregnancy. Similarly, RHD*993A,1136T occurs in a SCD patient and may impact transfusion recommendations. Together, these cases showcase the importance of molecular and serological investigation to categorize new RHD variants.
