We investigated the sample of a newborn male who presented with variable RH4 (c) phenotype, depending on reagents used. The patient had never received any red blood cell (RBC) transfusion. Given the high polymorphism of RHCE,1 we performed a RHCE study in order to settle his RH4 phenotype. Serologic testing was performed by standard methods using different monoclonal anti-RH4. The patient RBC were typed with anti-RH4 reagents by manual gel-test clone MS33 (BioRad, Hercules, CA) and by automation Ortho Vision, clone MS42 (QuidelOrtho, Raritan, NJ). Genomic DNA was extracted from white blood cells (NucleoMag, Macherey-Nagel, Hoerdt, France). RHCE genotyping was performed by: real time polymerase chain reaction (PCR) for C/c genotype, detection of c.340T typical of RHCE*ceJAL and c.667T typical of RHCE*ceMO (7500 Fast, Thermo Fisher Scientific, Waltham, MA) and Sanger sequencing of RHCE exons 1 to 10 with flanking intron regions (DX 3500, Thermo Fischer Scientific, Waltham, USA). RHD and RHCE microdeletions, microduplications and hybrid alleles were investigated by quantitative multiplex PCR of short fluorescent fragments (QMPSF).2 For additional insight about the impact of the variation found compared to a conventional cE RhCE protein, the recently published three-dimensional RhCE resolved by cryo-electronic microscopy (cryo-EM) was used to locate the predicted amino acid change,3 after adding the missing residues (not resolved by cryo-EM) and selecting the best model as previously described.4 The effect of the novel change on the RhAG2RhCE trimer was predicted using mCSM-membrane,5 a webserver dedicated to the analysis of the impact of single-point mutations on membrane protein stability. By serologic testing, the RBCs typed weakly RH:4 with Ortho vision instrument with mixed-field agglutination, and weakly (0.5+) by manual testing with BioRad anti-RH4. Patient RBCs also typed RH:1,2,-3,5 (D+C+E–e+) with no reduced reactivity noted. Real time PCR was consistent with heterozygous C/c, and RHCE*ceJAL and RHCE*ceMO alleles were absent. RHCE Sanger sequencing found a heterozygous single nucleotide c.872C>G change in exon 6, with no other change from conventional. The novel change located at rs374399829, is not listed in gnomAD v4.1.0. RHCE exon 2 sequencing found heterozygous c.150C>T, c.178C>A, c.201A>G, c.203A>G, c.307C>T, and c.335+135G>A changes, consistent with a C/c genotype. RHCE exon 5 sequencing found heterozygous c.676G>C consistent with E/e genotype and homozygous c.801+101G>A (intronic non coding variation with balanced allele frequency). Because of the variable reactivity of the RBC with anti-RH4 reagents and the discrepancy between the RH:-3 phenotype and the c.676G/C genotype, the novel change is presumed to be on a RHCE*cE allele, with conventional RHCE*Ce in trans. QMPSF analysis found two copies of both RHD and RHCE and was consistent with a C/c genotype, with no genetic recombination. The sequence has been submitted to GenBank (accession number: PX572761). The c.872C>G change in RHCE exon 6 is predicted to encode p.(Pro291Arg), located in the fifth RhcE extracellular loop, as shown in Figure 1. The change is not located in close proximity with the main antigenic determinants p.Pro103 (c) and p.Pro226 (E) but at the interface with RhAG. RhAG transmembrane glycoprotein is known to form a trimer with Rh proteins which in turn forms a cell membrane integrity stabilizing complex linked to the membrane skeleton via Ankyrin and protein 4.2.3 The c.872C>G change is predicted by mCSM-membrane to destabilize the complex formed by RhAG and RhCE (−0.022 Kcal/mol, the cut-off being at 0, suggesting only a modest effect). We report a novel RHCE allele with a single c.872C>G change, predicted to encode p.Pro291Arg and presumed to alter an extracellular residue in the fifth RhCE extracellular loop and destabilize the resulting RhAG2RhCE complex. Given the weak RH4 phenotype and RH3 discrepancy between phenotype and genotype (reported RH:-3 by serology, but with the presence of the variant usually associated with RH3, c.676C), we concluded to a novel RHCE*cE allele with an unknown risk of anti-RH3 and anti-RH4 formation. A similar variant in RHD exon 6, c.872 C>G (p.Pro291Arg, RHD*872G DEL41) has been reported in Swiss and German donors,6, 7 and no anti-RH1 has been reported for this extremely rare variant. Nevertheless, considering the extracellular location of the change, if the situation warrants prevention of alloimmunization (women of childbearing age, patients with sickle cell disease, repeated transfusion, or previous allo-immunization), it would be cautious to consider the resulting RH3 and RH4 antigens as potentially at risk of anti-RH3 and anti-RH4 formation and provide antigen-negative RBCs if possible. Outside of these circumstances, a specific transfusion protocol would not usually be recommended. Open access publication funding provided by COUPERIN CY26. 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.
Heng et al. (Wed,) studied this question.