RHD allelic variability in north Argentine

TRUCCO BOGGIONE, CAROLINA; SIPPERT, EMILIA; LUJÁN BRAJOVICH, MELINA ELIANA; GASPARDI, AC; LERI, MÓNICA; MATTALONI, STELLA MARIS; CASTILHO, LILIAN; COTORRUELO, CARLOS

Londres

Congreso; 25th Regional Congress of the ISBT; 2015

ISBT

Background: the molecular background of the D negative and D variant phenotypes shows substantial ethnic variability. The population of Argentine is considered to be a mixture of white Caucasian Europeans, Amerindians and Africans. It has been observed that the indigenous and African component is higher in the north region of the country. Aim: the aim of this study was to characterize the molecular background of D negative samples expressing C and/or E antigens (D-, C/E+) and D variant (Dvar) phenotypes in individuals from the city of Tucumán, located in North Argentine. Materials and Methods: 242 blood samples (D-, C/E+: n=187; Dvar: n=55) were selected for this study. The Rh status was determined by hemagglutination using specific monoclonal antibodies. The D antigen was evaluated with three different IgM anti-Ds and a blended anti-D. When an immediate spin?negative result was observed with the latter antiserum, the samples were tested by the indirect antiglobulin test. C, c, E and e antigens were also investigated. DNA samples from D-, C/E+ individuals were initially screened for the presence of the 5? untranslated region (UTR), intron 4 and the 3? UTR of the RHD gene using PCR strategies. Samples carrying RHD specific fragments and DNA samples from the D variant donors were studied by RHD exon scanning, PCR-SSP, PCR-RFLP, microarray and sequencing. Results: among the 187 D negative samples expressing C and/or E antigens, 40 (21.4%) carried RHD specific fragments. Hybrid alleles were found in 27 (67.5%) of these samples: 21 RHD-CE-Ds, 1 RHD-CE(4-9)-D, 4 RHD-CE(3-9)-D, 1 RHD-CE(4-7)-D2. In the remaining 13 samples, 6 (15.0%) harboured DEL variants: 5 RHD(46T>C), 1 RHD(IVS3+1G>A) and 7 (17.5%) carried null alleles: 6 RHD(581insG), 1 RHDΨ in a sample expressing the C antigen (probably a RHDΨce/dCe genotype). Twelve different alleles were responsible for the 55 Dvar samples studied: 12 (21.8%) weak D type 1, 7 (12.7%) weak D type 2, 7 (12.7%) weak D type 3, 12 (21.8%) weak D type 4, 5 (9.1%) DVI type 4,1 (1.8%) DFR-2, 1(1.8%) weak D type 5 and 1 (1.8%) DMH. Interestingly, 3 novel mutations were found in 9 samples: 1 (1.8%) RHD(763G>A), 1 (1.8%) RHD(764G>A) and 7 (12.7%) RHD(359C>A). Conclusions: the allelic variability found in North Argentine could be explained by the contribution of the Amerindian and African ethnicity to the genetic pool of the population. The finding of DEL alleles highlights the importance of genotyping D negative donors since these RBC units have the potential to cause anti-D alloimmunization in truly D negative patients. The understanding RHD allele repertoire in the analyzed population will help to develop reliable strategies in Blood Banks and prenatal RHD genotyping.