MOLECULAR BACKGROUNDS FOR THE Dce (R0) HAPLOTYPE

COTORRUELO C; MUNINI G; GARCÍA BORRÁS S; RACCA L; DI MÓNACO R; BIONDI C; RACCA A

Ciudad del Cabo - Sudáfrica.

Congreso; XXIX International Congress of the International Society of Blood Transfusion; 2006

International Society of Blood Transfusion

Introduction: the RH locus is composed by the highly homologous tandemly arranged RHD and RHCE genes which alleles segregate as haplotypes with frequencies varying in different ethnic groups. The RHD gene is flanked by the homologous upstream and downstream Rhesus boxes while a hybrid one is present in RHD deleted chromosomes. The detection of a hybrid Rhesus box in an RhD-positive individual denotes an RHD heterozygous status and helps to determine the Rh haplotype present. Cytosine 48 in RHCE exon 1, characteristic of the conventional RHC allele, and guanine 733 in RHCE exon 5, responsible for VS antigenicity, have been noted in many RHce alleles, mainly in Africans, but it was not clearly established whether they occurred in the same allele and/or cosegregate together with RHD. Aim of the study: to determine the molecular backgrounds for the Dce haplotype. Methods: blood samples from 148 white trios (father, mother and child) were studied. The Rh phenotype was performed by hemmaglutination. The RHD deleted allele was determined by a PCR strategy using primers that selectively amplify a segment of the hybrid Rhesus box. Serological and PCR inconsistencies were studied by a PCR-RFLP method that detects another polymorphic site of the hybrid Rhesus box. RHCE exon 1 G48C, RHCE exon 5 C733G and RHC intron 2 polymorphisms were analysed by allele specific PCR. The Rh haplotypes were determined considering (i) the RHD zygosity determined by Rhesus box analysis, (ii) the CcEe phenotype and (iii) the segregation analysis of Rh antigens in each trio. Frequencies were obtained analysing unrelated individuals (fathers and mothers, n=296). Results: the Dce haplotype frequency was 5.6%. Nucleotide 48C was found in all serologically positive RhC (n=201), in 9 Rhcc samples together with 48G and in 1 Rhcc phenotype in which 48G was absent. These Rhcc samples did not show the 109-bp insertion in intron 2 characteristic of the RHC allele. Six RhCc samples typed only 48C also indicating the presence of 48C in RHc alleles. The occurrence of 733G (VS+) was found in 12 samples that also typed 733C positive showing the heterozygosity of the C733G mutation. Segregation analysis showed that RHCE exon 1 cytosine 48 and RHCE exon 5 guanine 733 were present in RHce alleles that cosegregated with RHD forming Dce haplotypes and that both transversions are not frequently found in the same RHce allele. Of the 33 Dce haplotypes found, 16 (48.5%) had a C at position 48 [Dc(48C)e], 11 (33.3%) had a G at position 48 with a G at position 733 [Dc(48G)es], 5 (15.2%) had a G at position 48 [Dc(48G)e] and 1 (3.0%) had a C at position 48 together with a G at position 733 [Dc(48C)es]. Conclusions: The results obtained clearly show 4 molecular backgrounds for the Dce haplotype and reflect the contribution of African alleles to the genetic pool of the population under study. The molecular characterization of Dce, which represents the oldest Rh haplotype, and its frequency distribution, may develop a better understanding of the phylogeny of Rh haplotypes. ccotorru@fbioyf.unr.edu.ar