The low prevalence Rh antigen Be^a(Rh36) is associated withRHCE*ce662C>G in exon 5, which is predicted to encode Rhce 221Arg

Abstract

Background and Objectives The low prevalence antigen, Be a , is produced by a complex that also produces weak c, e and f (ce). We report here the molecular basis associated with Be a antigen expression. Materials and MethodsPeripheral blood samples from four Be(a+) probands were tested. Haemagglutination, gDNA extraction, PCR-based assays, reticulocyte RNA isolation, Rh-cDNA analyses, and sequencing were performed by standard procedures.Results RBCs from Probands 1 and 3 were D)C)E)c+e+, and from Probands 2 and 4 were D+C+E)c+ W e+. In proband 1, cDNA sequencing of RHCE revealed heterozygosity of nucleotide (nt) 662C ⁄ G in exon 5 of RHCE*ce. No other nucleotide changes were observed. As the 662C>G nucleotide change ablates a MscI restriction enzyme cleavage site, PCR-RFLP analysis was performed and the RHCE*ce nt 662C ⁄ G heterozygosity was detected on gDNA from the four probands and two children from both Proband 3 and Proband 4. ConclusionThe low prevalence Rh antigen, Be a , is associated with a single nucleotide change in exon 5 of RHCE*ce; that of 662C>G and this change is predicted to alter proline at amino acid position 221 of Rhce to arginine. The fundamental differences in the properties of these two amino acids may impose a steric and ⁄ or charge-related effect on the protein, and thereby provide an explanation for the weakened expression of c, e and f (ce) antigens in the Be a phenotype.

RHCE*cE734C allele encodes an altered c antigen and a suppressed E antigen not detected with standard reagents

“…Some examples of interactions between external loops providing weakening RH antigens at the RBC surface have been reported. In a recent publication, the RHCE*ce662G allele (Pro221Arg) was shown to be associated with RH36 antigen expression and with weakened expression of e antigen as well as c antigen, although the nucleotide substitution is found in Exon 5, which is known to code the seventh and eighth transmenbrane domains and the fourth extracellular loop 17 . In 2009, Bugert and colleagues 27 described a new RHcE allele associated with weak c antigen defined by a Leu303Gln substitution in the 10th transmembrane domain, adding weight to the argument in favor of c reactivity being dependent on interaction between external loops of RhCE.…”

“…The molecular bases of RHCE variants are generally gene rearrangements or single or multiple nucleotide substitutions resulting either in weak e antigen expression ( RHCE*ce48C ) 11 or partial e antigen expression ( RHCE*ceMO 9 or RHCE*ceAR 10,12 ). These events may also lead to the loss of a high‐prevalence RhCE antigen (RH18 for RHCE*ceAR , 10 RH19 for RHCE*ceMO 9 and RHCE*ceRA , 13 RH26 for RHCE*ce286A 14 ) or in the production of a low‐prevalence RhCE antigen (RH32 for R N , 6 RH48 for RHCE*ce s 340T , 15 RH55 for RHCE*ce286A , 16 RH36 for RHCE*ce667G 17 ).…”

A novel RHCE*ce 48C, 733G allele with Nucleotide 941C in Exon 7 encodes an altered red blood cell e antigen

“…Reverse transcription was carried out with gene‐specific RHD and RHCE primers as published previously 7 and reverse transcriptase, according to manufacturer's instructions (SuperScript III First Strand Synthesis SuperMix, Invitrogen). PCR amplification was carried out as described previously with primers cRHx1F and cRHx5R to amplify Exons 1 through 4 and cRHx4F and cRHx10R to amplify Exons 5 through 10 in RHD and RHCE cDNA 7 using premixed solution (HotStarTaq Master Mix Kit, Qiagen). PCR products were checked for purity on agarose gels, cleaned using ExoSAP‐IT (USB Corporation) according to manufacturer's instructions and directly sequenced (Genewiz, Inc.).…”

“…5,6 RNA extraction and Rh cDNA cloning and sequencing RNA was isolated from reticulocytes using standard techniques (TriZol and PureLink Micro-to-Midi Total RNA purification system, Invitrogen). Reverse transcription was carried out with gene-specific RHD and RHCE primers as published previously 7 and reverse transcriptase, according to manufacturer's instructions (SuperScript III First Strand Synthesis SuperMix, Invitrogen). PCR amplification was carried out as described previously with primers cRHx1F and cRHx5R to amplify Exons 1 through 4 and cRHx4F and cRHx10R to amplify Exons 5 through 10 in RHD and RHCE cDNA 7 using premixed solution (HotStarTaq Master Mix Kit, Qiagen).…”

Rh and RHAG Blood Group Systems

“…Anti -Be a has been implicated in several cases of severe HDFN [433,455 -457] . Be a is encoded by an RHCE * ce allele that produces slightly weakened c, e, and ce and contains 662C > G in exon 5 encoding Pro221Arg in or close to the extracellular vestibule [433] .…”