(P-IG-4) A Novel RHD Allele Combining c.455A >C (DNT Variant) with c.1154G >C (Weak D Type 2 Variant)

Background/Case Studies: Weak D type 2 (c.1154G >C, p.Gly385Ala) expresses a very weak RhD phenotype, is usually linked to cE and European ancestry, and is managed as D+ with nil risk of clinically significant alloanti-D. The c.455A >C (p.Asn152Thr) gene conversion from RHCE is a key variant in the DIVa partial RHD phylogenetic cluster. Isolated c.455C comprises RHD*38 (DNT) which has normal-strength RhD expression and is associated with anti-D (Shin DW, Am J Clin Pathol 2024;161:111-4).

Study

Design/Methods:

Routine RBC RhD typing was by automated microplate direct agglutination (MDA) and antibody screen was by solid phase red cell adherence (Neo). Equivocal MDA RhD typing was investigated with automated antiglobulin RhD typing, tube RhD and CcEe typing (Gamma-clone), and RHD BeadChip microarray genotyping (all Immucor, Werfen, Norcross, GA). RHD deletion was sought by polymerase chain reaction for the hybrid Rhesus box. Sanger sequencing was performed on RHD exons 1-10 and flanking intron regions.

Results/Findings: A 33-year-old White non-Hispanic woman from the midwestern United States (US) with southern European ancestry presented in early first pregnancy. Her RhD typings were equivocal by Series 4 and negative by Series 5 in MDA, 4+ in antiglobulin and 3+ mixed-field in tube. The CcEe phenotype was cEe. The antibody screen was negative. The RHD BeadChip showed hemi- or homozygosity (hxm) for c.1154G >C and called the result weak D type 2. However, inspection of probe results also revealed hxm for c.455A >C. The hybrid box was detected, indicating hemizygosity. Sanger sequencing showed hxm for c.455C, c.1154C, and type-2-linked c.1154-31C >T.

Conclusions: RHDTo our knowledge the RHD*455C,1154C allele has not been reported previously. In our transfusion service, weak D type 2 cases (n=3) were D-negative in MDA and found in tube confirmatory typing (1+). Partial RHD alleles in the DIVa cluster have c.455C with other variants and were usually linked to ce haplotypes and African ancestry. However, isolated c.455C DNT was reported in diverse US Black, German White and Korean Asian ancestries and linked to Ce or cE. Our case’s linkage to cE and c.1154-31C >T suggest c.455A >C microconversion in a weak D type 2 allele. Her RhD phenotype strength was intermediate between weak D type 2 and the full-strength RhD of DNT. Considering the published reports of anti-D in DNT, we recommended management as D-negative for RBC transfusion and Rh immunoprophylaxis. The analogous RHD*62 (DNT(V270G)) allele had c.455C with weak D type 1 variant c.809T >G (RhesusBase, unpublished), but its phenotype is unknown. Our case illustrated the importance of reviewing RHD BeadChip probe results for unexpected findings. The BeadChip call ignored c.455A >C and the interpretation as weak D type 2 may pose risk for anti-D alloimmunization.