Molecular polymorphism of the RH locus in D negatives Argentineans

LUJÁN BRAJOVICH, MELINA; TRUCCO BOGGIONE, CAROLINA; GARCÍA BORRÁS, SILVIA; BIONDI, CLAUDIA; RACCA, AMELIA; COTORRUELO, CARLOS

El Cairo

Congreso; XIXth Regional Congress of the ISBT; 2009

International Society of Blood Transfusion

Background: the RH locus is composed by the RHD and RHCE genes that encode the RhD and RhCcEe polypeptides respectively. Both genes are highly homologous and form a cluster with a tail-to-tail orientation. The RhD negative phenotype in Caucasians is mainly caused by a complete deletion of the RHD gene. However, the presence of significant portions of the RHD gene in RhD negative individuals has been reported in different ethnic groups. These alleles confound DNA typing because they may lead to false positive results. Aims: the purpose of this study was to analyse the frequency of RhD negative individuals carrying a silent RHD allele in the population of Rosario, the third largest city of Argentina. Materials and Methods: blood samples from 440 self-identified white RhD negative blood donors were studied. The Rh phenotype was performed by hemmaglutination. The presence of the D antigen was further analysed by the indirect antiglobulin test. DNA samples were initially screened using a multiplex PCR strategy that amplifies intron 4 of the RHCE (1238 bp) and RHD (587 bp) genes and the 3? unstranslated region of the RHD gene (229 bp). PCR products were analysed in 2% agarose gel electrophoresis. Samples carrying RHD specific fragments were further studied by RHD exon scanning with PCR SSP and PCR RFLP strategies, PCR SSP for the C733G mutation and RHD specific PCR. Results: in the 440 RhD negative samples studied, 27 (6.14%) were C positive, 7 (1.59%) E positive and 3 (0.68%) C and E both positive. Molecular studies showed that 7 samples carried RHD specific fragments. In 3 samples (C positive, E negative) we detected RHD/CE hybrid genes: 2 RHD-CE(2-7)-D which also had the C733G mutation and 1 RHD-CE(3-7)-D. The RHD allele was detected in 2 samples (C and E negative). In the remaining 2 samples (1 C negative, E positive and 1 C and E both negative), a normal RHD exon scanning was found. Conclusions: we have found the presence of RhD negative / RHD positive individuals in our population. The d(C)ceS (r?s) haplotype, classically found in Africans, has been detected in our population but contrary to previous reports it was associated with the RHD-CE(2-7)-D hybrid allele. The African influence is also reflected by the presence of the RHD allele. These findings highlight the importance of the study of the molecular polymorphism of the RH locus so as to develop reliable DNA typing strategies.