CHARACTERIZATION OF A RHCE NULL ALLELE IN A CAUCASIAN DONOR

PRINCIPI, CINTIA; LUJÁN BRAJOVICH, MELINA ELIANA; TRUCCO BOGGIONE, CAROLINA; MATTALONI, STELLA MARIS; ENSINCK, MARÍA ALEJANDRA; BIONDI, CLAUDIA; COTORRUELO, CARLOS

Congreso; 37th International ISBT Congress; 2022

ISBT

Background: the Rh system is genetically controlled by the homologous RHD and RHCE genes. Rearrangementsbetween both genes might generate null alleles that are responsible for the lack of reactions of red blood cells withantisera that define one or more of the Rh antigens.Aims: 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 antigenexpression was identified. We studied the D and CE status with commercial micro-column gel cards. The expression ofthe 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-negativecells were tested as controls. Genomic DNA was isolated from peripheral blood with a commercially available purificationkit. Quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF) of RHD and RHCE genes was performed todetermine presence/absence of all RH exons. Further PCR-SSP strategies were also used to study RHCE specificpolymorphisms in exon 1 (nt 48), exon 2 (nt 201), intron 2 (ins 109pb), exon 3 (nt 383), exon 9 (nt 1193) and the 3’ UTRregion. PCR-RFLP analysis with Bcl I, Taq I, Rsa I and Alu I endonucleases were performed as well to analyse exon 4 (nt594), exon 5 (nt 697), exon 6 (nt 932) and exon 7 (nt 974) polymorphisms, respectively. RHD zygosity was investigatedby 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-eantisera. The flow cytometric results demonstrated 48% and 52% overexpression of the D antigen with anti-D clones MS201 and ESD1M respectively, compared to R1R2 cells (standard positive control). Accordingly with serologic results, noantigen expression was detected with anti C, c, E and e monoclonal antibodies. QMPSF studies allowed the identificationof all RHD exons and showed the absence of RHCE exons 3, 4, 5, 6 and 7. This analysis also showed two copies ofRHCE exon 1 (cytosine 48) and two copies of RHCE*c exon 2. PCR-SSP and PCR-RFLP studies confirmed the previousfindings. Zygosity analysis suggested a homozygous status of the RHD gene.Summary / Conclusions: the results obtained by serologic and molecular methods suggest that the D-- phenotyperesulted 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 mayhave occurred in a R0 haplotype carrying the RHCE*ce.01 allele. The presence of RHD regions over most of its length inthe recombinant structure found may account for the lack of expression of CE antigens and the overexpression of the Dantigen. This is an unusual phenotype in the Argentinean population.