The human red blood cells (RBCs) carry 48 blood group systems, among which the Rh system is one of the most complex. RH antigens are encoded by two closely linked genes, RHD and RHCE. The International Society of Blood Transfusion Red Cell Immunogenetics and Blood Group Terminology Working Party catalogs for the RHCE gene more than 400 different alleles (https://blooddatabase.isbtweb.org/system/RH). We investigated two blood donors of European ancestry from the same family (father and daughter, respectively, Donor 2 and Donor 1) whose RBC typed D + C-E + wc + e+. The samples were sent to our laboratory to resolve the discrepancy of E antigen expression. Serologic testing was performed by standard hemagglutination techniques with multiple anti-E: P3x234 + MS258, 906 clone from Diagast (Loos, France), and MS258 + MS80 clone from Werfen (Georgia, USA). DNA was extracted from whole blood using EZ 1 & 2 DNA Blood Kit 350 μL (Qiagen, Venlo, Netherland) according to the manufacturers' instructions. Genotyping was performed by real-time PCR for c.602G>C (RHCE*cEIV) and c.676G>C (E/e polymorphism) (ABI 7500 Fast Real-Time PCR System Thermofisher, Les Ulis, France) and Sanger sequencing of RHD and RHCE exons 1–10 and flanking intron regions (3500Dx genetic analyzer, Thermofisher, Les Ulis, France).1, 2 Zygosity was determined by quantitative multiplex PCR of short fluorescent fragments (QMPSF) as previously reported.3 By serologic testing, the two donors were negative or weekly reactive (2+) with anti-E clone from Diagast, but positive (3+ and 4+) with anti-E clone from Werfen (Table 1). By Real-time PCR the results were wildtype for c.602G and heterozygous for c.676G>C. The RHCE sequencing analysis led to the following results: For the two samples, a heterozygous c.689G>C was found in RHCE exon 5, together with the E/e polymorphism (c.676G>C). In RHCE exon 2, the c polymorphisms were found (c.150 T>C, c.178A>C, c.201G>A, c.203G>A, and c.307 T>C), and no other variant in other exons. The single nucleotide variant (SNV) c.689G>C is predicted to encode a serine to threonine substitution at amino acid position p.230. Sequencing did not reveal any variant in RHD, and QMPSF showed the sample to be hemizygous for RHD. Based on the D + C-E + wc + e+ phenotype, the novel c.689G>C variant can be assigned to the RHCE*cE allele, likely within an R2 (DcE) haplotype. An allele with the same variant on a different background, RHCE*02.15 has been reported in a German donor following the observation of weak or absent reactions with some anti-e. The donor's RBC typed D + C + E + c + e+w, with no alteration of E expression.4 In this work, we describe a novel RHCE*cE allele provisionally designated RHCE*03.38 by ISBT that alters the expression of the E antigen in two, related individuals of European descent. The amino acid substitution at p.230 is predicted to be located at the fourth external loop.5 Based on this extracellular position and the discrepant serology results (RBC nonreactive with at least one anti-E reagent), we recommend to treat this new allele as responsible for a partial E phenotype. The sequence was deposited in Genbank under accession number PX273697 and provisionally designated RHCE*03.38 by ISBT. The c.689G>C SNV is not in dbSNP nor in gnomAD. Open access publication funding provided by COUPERIN CY26. 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.
Braga et al. (Sat,) studied this question.