New <i>RHCE*CE(c.382G &gt; a)</i> allele in patients of Asian ancestry

The International Society of Blood Transfusion Red Cell Immunogenetics and Blood Group Terminology Working Party1 catalogs for the RH blood group system more than 400 different alleles in populations from various geographic origins. Three patients (P1, P2, and P3) and one donor (D1) of Asian ancestry, all female, were referred for genotyping to resolve the serologic weak E or weak e typing. P1, P2 were women of childbearing age whose red blood cells typed D+ C+ E+wc-e + and were referred to investigate the E weak phenotype. P2 was pregnant. P3 and D1 (also of childbearing age), whose red cells typed D+ C+ E+c- e+w, were referred to investigate the e weak phenotype. Automated or manual serologic typing for D, C, E, c and e was performed by standard hemagglutination techniques using commercially available reagents from QuidelOrtho Clinical Diagnostics (Raritan, New Jersey, USA), Diagast (Loos, France), Grifols (Barcelona, Spain), Bio-Rad (Hercules, CA, USA), Immucor (now Immucor-Werfen, Georgia, USA) or Eurobio Scientific (Les Ulis, France). Genomic DNA was isolated from whole blood with MagnaPure Compact Nucleic Acid Isolation Kit I (Roche, Meylan, France) or NucleoMag (Macherey-Nagel, Hoerdt, France) according to the manufacturers' instructions. Genotyping was performed for all samples using the RHCE BeadChip array (BioArray Solutions/Immucor-Werfen, Warren, New Jersey, USA) and Sanger sequencing (RHCE exons 1 to 10 for P1, P3, and D1, RHCE exon 3 and 5 for P2, and RHD exons 1 to 10 for P1–3500 Dx genetic analyzer, Thermo Fisher), as previously described.2, 3 Allele specific amplification and sequencing was performed for D1 to determine the cis/trans relationship for the detected single nucleotide variations (SNV) (Figure 1). The red cells from P1 and P2 were very weakly reactive with anti-E (clone 906 - Diagast) and weakly reactive using MS260 (Grifols) or C2 (Ortho) anti-E (Table 1). The red cells from P3 were very weakly reactive using a blend of anti-e: M16-MS21-MS63 (Ortho and Bio-Rad) or P3GD512-MS63 (Diagast). Furthermore, the reactivity with anti-E was weak with clone 906. The red cells from D1 were very weakly reactive with P3GD512-MS63 anti-e blend, and reactive (strength of reaction was not recorded) with HS128-MS21 (Diagast), M16-MS21-MS63 (Immucor), and MS62-MS69 (Eurobio Scientific) anti-e blends. Results for RH3 were not graded as no ambiguity was recorded for D1 with anti-E clones 906 and P3x234-MS258 (Diagast). The RHCE Beadchip and sequencing analysis lead to the following results: for all samples, a novel, heterozygous c.382G > A was found in RHCE exon 3, together with the E/e and C polymorphisms. For P1 and P2, no other change was found, and because of the D+ C+ E+ wc-e + phenotype, this mutation can be assigned to the RHCE*CE allele: RHCE*CE382A. In P3, an additional heterozygous c.1007G > A predicted to encode p.Gly336Asp was found in exon 7. Because of the D+ C+ E+ c-e + w phenotype, we assume the association of the novel RHCE*CE382A allele in trans with the previously reported allele RHCE*Ce1007A (RHCE*02.24) for P3. In D1, an additional heterozygous c.375C > G predicted to encode p.Ile125Met was found in exon 3. This known allele RHCE*Ce375G (RHCE*02.27), which explains the serologic weak e phenotype, is associated in trans with the RHCE*CE382A allele in D1. This was confirmed in D1 by allele specific amplification and sequencing (Figure 1). RHCE was not fully sequenced for P2, which is the most recent sample, because the novel allele presented in this report had already been fully sequenced for the three other samples and c.382A could be considered the cause of the weak E phenotype. The c.382G > A SNV is found as rs369664408 with 0.00008912 allele frequency in East Asians (gnomAD v.4.1.0).4 The novel c.382G > A change is predicted to encode p.Gly128Ser in the 4th transmembrane domain of the RhCE protein based on a recent protein model of RhD.5 In this work, we describe a novel RHCE*04 allele (RHCE*CE382A) that alters the expression of the E antigen in individuals of Asian descent (D1 was of Cambodian descent, while patients were of unspecified Asian descent). The amino acid involved (p.Gly128) has been associated previously on a RHCE*cE background with weak E phenotype: RHCE*03.15.01, RHCE*03.15.03, RHCE*03.28, and RHCE*03.32.6-8 D1 and P3 were not referred because of weak E typing, but because of weak e typing, and c.382A is an incidental finding. We hypothesize that the absence of weak E phenotype in these samples may be caused by the lower number of RhCe protein due to the RHCE*Ce variant alleles in trans. P2 was pregnant at the time of the investigation and delivered a neonate whose red cells typed D+ C+ E-c+ e+. D1 had a history of a least one pregnancy. None of the probands reported here had a history of transfusion or positive antibody screening test. Very few RHCE*CE alleles have been reported to date, and the novel RHCE*CE382A allele has been provisionally assigned RHCE*04.03 designation by ISBT and sequence was deposited in Genbank under accession number PQ212705. Caution for this rare allele would compel to consider the C and E expressed by RHCE*CE382A as partial until proven otherwise and transfuse P1 and P2 with E-blood. However, the compound heterozygosity with rare alleles also potentially expressing partial antigens in D1 and P3 create a rare phenotype with potentially partial E and partial e. If these individuals required transfusion, unit selection should be considered on a case-by-case basis.