A serologic weak D caused by a novel <i>RHD</i> allele with single nucleotide c. <scp>209G</scp> &gt;C variation predicted to encode p. <scp>Arg70Pro</scp>

We investigated the sample of a 34-year-old French female of Albanian ancestry during the 28th week of amenorrhea of her first pregnancy. The patient presented with a serologic weak D phenotype and had never received red blood cell (RBC) transfusion. RhD is a transmembrane RBC protein. Given the considerable polymorphism of RHD,1 the D antigen immunogenicity and the hemolytic transfusion reactions and hemolytic disease of the fetus and newborn danger due to anti-D,2 genotyping RHD is essential to dispense the most appropriate transfusion and obstetrical guidance. Per national guidelines, the sample was sent for RHD genotyping to investigate the weak D phenotype. Serologic testing was performed by using different monoclonal anti-D. The patient RBCs were typed by automation [Microplate erythrocyte magnetization with Qwalys 3 EVO instrument, clone P3X61 (Diagast, Hoerdt, France), gel test with IH-500, clones LHM 50/3 (LDM1)/TH-28/RUM-1 and LHM50/20 (LDM3)/175-2 (BioRad, Hercules, CA) and Ortho Vision, clone D7B8 (QuidelOrtho, Raritan, NJ)] and by manual gel card testing, clone P3X61 (Grifols, Barcelona, Spain). Genomic DNA was extracted (NucleoMag, Macherey-Nagel, France) and RHD genotyping was performed by: real-time polymerase chain reaction (PCR) for c.809G typical of RHD*weak D type 1 and c.8G typical of RHD*weak D type 3 (7500 Fast, Thermo Fisher Scientific, Waltham, MA), RHD Beadchip (BioArray Immucor-Werfen, Warren, NJ) and Sanger sequencing of RHD exons 1 to 103 and RHCE exon 24 with flanking intron regions (DX 3500, Thermo Fischer, USA). The previously reported three-dimensional homology model5 was used to locate the predicted amino acid change compared to a conventional RhD protein. By serologic testing, the RBCs typed weak D with Qwalys (score 47, positive very weak), Ortho vision (3+) and IH-500 (3+) instruments and in manual testing (3+). Patient RBCs were also typed C + E − c + e + (RH: 2, −3, 4, 5) with no reduced reactivity noted. Real time PCR for weak D type 1 and weak D type 3 and BioArray RHD Beadchip found no changes from conventional. RHD sequencing found a homozygous single nucleotide c.209G>C change in exon 2. No changes were found in RHD exons 1 and 3 to 10. RHCE exon 2 sequencing found heterozygous c.150 C>T, c.178 C>A, c.201 A>G, c.203 A>G, c.307 C>T and c.335+135G>A changes, consistent with a C/c genotype. The c.209G>C change in RHD exon 2 is predicted to encode p.Arg70Pro amino acid substitution and to be located in the first RhD intracellular loop,5 as shown in Figure 1. This nucleotide change is not listed in gnomAD (although rs142925159 is listed for c.209)8 or the ISBT table of RHD alleles.9 The sequence was submitted to GenBank (accession number PV691508). We report a novel RHD allele with a single c.209G>C change, predicted to encode p.Arg70Pro and presumed to alter an intracellular residue in the first RhD intracellular loop. The risk of anti-D formation is unknown, as this allele has never been reported before,10 including in the Albanian population.6 A similar variant c.209A (p.Arg70Gln, RHD*weak D type 85) without anti-D has been reported.7 Nevertheless, even if anti-D formation seems unlikely because of the location of the amino acid, it may be prudent to consider the resulting D antigen as potentially at risk of anti-D formation in certain clinical settings, including women of childbearing age. It may be preferable to transfuse D negative RBC and inject anti-D gamma globulin in case of pregnancy. The patient was managed as D-negative and offered anti-D gamma-globulin. Her antibody screen was negative and her pregnancy was uneventful. A family study could not be performed. The newborn did not require a blood sample and no blood group was performed. The authors have nothing to report. The authors declare no conflicts of interest. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.