Novel <i>RHD*49C</i> allele identified in a prenatal patient

The Rh blood group system includes to-date 56 antigens, with the D antigen being the most immunogenic.1 More than 500 RHD alleles are listed on the ISBT website,2 with many encoding altered D antigens that may present with variable agglutination strengths, partly dependent on methodology and reagent. Without a robust policy, variable D expression may lead to inconsistent interpretation, complicating Rh immune globulin (RhIG) administration decisions, and causing inappropriate selection of red blood cells (RBCs) for transfusion.3 Pregnant or transfused patients with altered D expression due to loss of D epitopes, known as a partial or variant-D, are at risk of developing anti-D in cases when the fetus or transfused RBC expresses the missing epitopes. Women of childbearing potential with partial D phenotype should be treated as D-negative and as candidates for RhIG. Partial blood donors with partial D should be treated as D-positive to prevent alloanti-D immunization in D-negative recipients. The AABB released the “Joint Statement of Phasing-In RHD Genotyping for Pregnant Women and Other Females of Childbearing Potential with a Serological Weak D Phenotype” to address partial/weak D expressions and the rationale for RHD genotyping to resolve weak D phenotypes in pregnancy.4, 5 Utilizing RHD genotyping in cases of serological discrepancy/uncertainty in females of childbearing age may guide product utilization, perinatal care, and laboratory strategy on reporting D antigen status. In the United States, one 300 μg dose of RhIG is recommended for D-negative women at 28 weeks of gestation and within 72 h of birth when the newborn is confirmed to be D-positive.6 It is also recommended a prenatal patient be tested at the first prenatal visit for ABO/Rh and screen for IgG antibodies to RBCs.7 The hospital transfusion service's D-typing policy states the RBCs of females of childbearing potential (less than 50 years old) must demonstrate by automated method a reaction strength of ≥2+ to be reported as D-positive. If the reaction strength is <2+, tube testing is performed. If the tube testing reaction strength at immediate spin is <2+, the sample is reflexed for RHD genotyping and the patient is treated as D-negative and is a candidate for RhIG pending genotyping results. The hospital transfusion service's D-typing policy for neonates is any positive reaction strength is considered D-positive and is an indication for RhIG in D-negative or known partial D women. In this case report, a 24-year-old, G1P0 Midde Eastern female presented with variable D reaction strength, prompting further investigation. Automated microplate hemagglutination method was performed on ECHO Lumena for ABORH testing using D-antisera series 4 (D4) and series 5 (D5) blends (Werfen, Norcross, GA, USA) for samples collected at 16 weeks gestation and at delivery (sample 1 and sample 2, respectively). Automated solid phase method was performed on the ECHO for weak D testing using D4 on Sample 2. The tube method used D4 and D5 for both samples and Ortho BioClone (San Diego, CA, USA), Bio-Rad Seraclone (Hercules, CA, USA), and AliveDX ALBAclone (Chicago, IL, USA) anti-D for Sample 1. Tube method was tested at immediate spin, and at the antihuman globulin phase for the serologic weak D testing for Sample 2. Genomic DNA was extracted (QIAGEN, Valencia, CA, USA) from buffy coat. RHD genotyping methods included the RHD BeadChip (Werfen, Warren, NJ, USA), hybrid Rhesus box assay,8 and Sanger sequencing of RHD exons 1–10 and flanking intron regions.9 Table 1 summarizes the serology results. Sample 1 RBCs displayed weak reactivity with Werfen Anti-D in tube and automated methods, and strong reaction strengths in tube with Ortho, Bio-Rad, and AliveDx Anti-D. Sample 2 RBCs had stronger reactivity with Werfen Anti-D in automated and tube methods. The RHD BeadChip reported indeterminate calls (“Ax”) for the assays that interrogate c.48G>C and c.48G>A. The sample was hemizygous for RHD, as determined by amplification of hybrid Rhesus box. Sanger sequencing identified c.49G>C (p.Ala17Pro) in RHD exon 1 (GenBank #: PQ124103) (Figure 1). The variant is not listed on the database gnomAD v.4.1.010 but similar ones are there (c.49G>A and c.49G>T, rs762631049). We report a novel RHD allele with c.49G>C (p.Ala17Pro) (NM_016124.6:c.49G>C; NP_057208.2) in a Middle Eastern pregnant woman. The proximity of c.49 to c.48 may affect binding of the c.48G, c.48C, and c.48A BeadChip probe(s), resulting in the indeterminate “Ax” calls. The encoded Proline at position p.17 is predicted to reside in the transmembrane region, potentially affecting the RhD protein's ability to integrate into the membrane. The variable reactivity observed in tube for D4 and D5 with samples 1 and 2 is potentially due to the length of time from date of collection to testing. Sample 1 was tested in tube 6 days after date of collection and Sample 2 was tested on the day of collection. The impact of the variant on the exposed D antigen and associated risk for alloanti-D is unknown; therefore, the patient was treated as D-negative and administered RhIG. The patient gave birth to a healthy baby, whose RBCs displayed strong reactivity with D4, and a negative Direct Antiglobulin Test (DAT). The baby's sample was not sent for RHD genotyping. The patient had no history of antibodies, and anti-D was not detected in pregnancy. Although Rh-prophylaxis (RhIG) was administered at 29 weeks gestation, the patient had a negative antibody screen at delivery. Interestingly, no maternal-fetal bleeding events were documented during the pregnancy and the patient's DAT was negative. The patient will continue to be treated as D-negative and a candidate for RhIG. The authors have disclosed no conflicts of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request.