Characterization of a RHCE null allele in a Caucasian donor

PRINCIPI C; LUJÁN BRAJOVICH M; TRUCCO BOGGIONE C; MATTALONI S; ENSINCK A; BIONDI C; COTORRUELO C

Kuala Lumpur

Congreso; 37th International Congress of the International Society of Blood Transfusion; 2022

International Society of Blood Transfusion

Background: the Rh system is genetically controlled by the homologous RHD and RHCE genes. Rearrangements between both genes might generate null alleles that are responsible for the lack of reactions of red blood cells with antisera that define one or more of the Rh antigens.Aim: the aim of this study was to investigate the molecular genetic basis of an Argentinean donor with no detectable C, c, E and e antigens by standard serologic techniques.Methods: peripheral blood sample of the donor was referred to our reference laboratory since no C, c, E or e antigen expression was identified. We studied the D and CE status with commercial micro-column gel cards. The expression of the D, C, c, E and e antigens was also analysed by flow cytometry with the following IgM clones: anti-D MS-201/ESD1M, anti-C MS-24, anti-c MS-33, anti E MS-260/MS80+MS258 and anti-e MS-16/MS-21/MS-63. R1R2 and antigen-negative cells were tested as controls. Genomic DNA was isolated from peripheral blood with a commercially available purification kit. Quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF) of RHD and RHCE genes was performed to determine presence/absence of all RH exons1. Further PCR-SSP strategies were also used to study RHCE specific polymorphisms in exon 1 (nt 48), exon 2 (nt 201), intron 2 (ins 109pb), exon 3 (nt 383), exon 9 (nt 1193) and the 3’ UTR region. PCR-RFLP analysis with Bcl I, Taq I, Rsa I and Alu I endonucleases were performed as well to analyse exon 4 (nt 594), exon 5 (nt 697), exon 6 (nt 932) and exon 7 (nt 974) polymorphisms, respectively. RHD zygosity was investigated by PCR-RFLP.Results: red blood cells were positive for the anti-Ds tested and failed to react with anti-C, anti-c, anti-E and anti-e antisera. The flow cytometric results demonstrated 48% and 52% overexpression of the D antigen with anti-D clones MS-201 and ESD1M respectively, compared to R1R2 cells (standard positive control). Accordingly with serologic results, no antigen expression was detected with anti C, c, E and e monoclonal antibodies. QMPSF studies allowed the identification of all RHD exons and showed the absence of RHCE exons 3, 4, 5, 6 and 7. This analysis also showed two copies of RHCE exon 1 (cytosine 48) and two copies of RHCE*c exon 2. PCR-SSP and PCR-RFLP studies confirmed the previous findings. Zygosity analysis suggested a homozygous status of the RHD gene.Conclusions: the results obtained by serologic and molecular methods suggest that the D-- phenotype resulted from a macroconversion event between the RHD and RHCE genes leading to a rare RHCE(1-2)-D(3-7)-CE(8-10) hybrid allele. The detection of two copies of both RHCE exon 1 and exon 2 by QMPSF also suggests that this event may have occurred in a R0 haplotype carrying the RHCE*ce.01 allele. The presence of RHD regions over most of its length in the recombinant structure found may account for the lack of expression of CE antigens and the overexpression of the D antigen. This is an unusual phenotype in the Argentinean population.