Serological and molecular studies of a D-- phenotype

LUJÁN BRAJOVICH M; TRUCCO BOGGIONE C; MATTALONI SM; GARCÍA BORRÁS S; RUCCI A; BERISVIL C; BIONDI C; COTORRUELO C

Londres

Congreso; 25th Regional Congress of the International Society of Blood Transfusion; 2015

International Society of Blood Transfusion

Background: the Rh system is genetically controlled by the homologous RHD and RHCE genes that encode the RhD and RhCcEe polypeptides respectively. Deletions, point mutations and rearrangements between both genes are responsible for the great polymorphism of this system. In rare cases red blood cells totally fail to react with antisera that define one or more of the Rh antigens. Aim: the aim of this work was to study a sample with no C, c, E or e antigen expression. Materials and Methods: a peripheral blood sample of a patient was referred to our reference laboratory because of the presence of a panreactive antierythrocyte antibody. Extended red blood cell phenotype analysis was performed using tube and microplate techniques. The expression of the D, C, c, E and e antigens was also determinated by flow cytometry. An IgM anti-D (clone MS-201), IgM anti-D (clone ESD1M), anti-C (clone MS24), anti-c (clone MS33), anti-E (clone MS80 + MS258) and anti-e (clones MS16 + MS21 + MS63) were used. R1R2 and antigen-negative cells were tested as controls. PCR-SSP strategies were used to study RHCE specific polymorphisms in exon 1 (nt 48), exon 2 (nt 201), intron 2 (ins 109pb), exon 3 (nt 383), exon 5 (nt 676), 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 to study exon 4 (nt 594), exon 5 (nt 697), exon 6 (nt 932) and exon 7 (nt 974) respectively. A Taq I site present in intron 1 and a Pst I site RHC/c-associated polymorphism in intron 2 were also analysed. Results: serological studies showed an IgG 37ºC-reactive alloantibody. Propositus? 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 suggesting a D-- phenotype with anti-Rh17. The flow cytometric results demonstrated 43% and 47% overexpression of the D antigen with anti-D clones MS-201 and ESD1M respectively, compared to R1R2 cells (standard positive control). Accordingly with serological results, no antigen expression was detected with anti C, c, E and e monoclonal antibodies. Molecular analysis revealed the absence of RHCE sequences 3? of the 4.2 Kb homology region encompassing exon 2. No RHCE specific polymorphisms were found in exons 3, 4, 5, 6 and 7 while RHCE specific nucleotides were detected in exon 9 and the 3?UTR. Therefore, the 3? breaking point of this rearranged allele may occur within intron 7 and intron 8. Conclusions: molecular studies confirmed the serological findings and suggest that the D-- phenotype resulted from a macroconversion event between the RHD and RHCE genes involving a segmental replacement of RHCE sequences with homologous RHD, generating a hybrid allele. The presence of RHD regions over most of its length in the recombinant structure found may account for the overexpression of the D antigen. To our knowledge, this is first description of this unusual phenotype in an Argentinean patient.