Oral Presentation: Molecular structures identified in serologically D negative samples of an admixed population

TRUCCO BOGGIONE C; LUJáN M; TARRAGO M; RACCA A; MUñIZ DIAZ E; NOGUES N; COTORRUELO C

Amsterdam

Congreso; 23rd Regional Congress of the International Society of Blood Transfusion; 2013

International Society of Blood Transfusion

Backgound: The D negative phenotype is caused by the complete deletion of the RHD gene in Caucasians. However, a plethora of allelic variants have been described among D negative individuals from different ethnic groups. The population of Argentina is composed predominantly by Caucasian Europeans admixed with Amerindians and Africans. Aim: The aim of this work was to study the molecular basis of the D negative phenotype in individuals from the city of Rosario. Materials and Methods: 1314 D negative blood samples were randomly selected. The D antigen was evaluated by hemagglutination with a blended anti-D (IgM TH-28/IgG MS-26). When an immediate spin?negative result was observed, the samples were tested by the indirect antiglobulin test. C, c, E and e antigens were also determined. DNA samples 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. RHD zygosity of all samples containing RHD specific sequences was evaluated by Rhesus box analysis by PCR-RFLP. D negative/RHD positive samples were studied by PCR-SSP, microarray and sequencing. Results: Among the 1314 D negative samples, 1200 were rr phenotypes while 114 carried C and/or E antigens. RHD specific amplifications were found in 27 samples (2.1%). One hybrid Rhesus box was detected in all D negative/RHD positive samples suggesting a hemizygous status. The RHDØ allele was found in 8 (0.7%) of the 1200 rr samples. DEL and null variants were detected in 19 (16.7%) of the 114 D negative phenotypes expressing C and/or E antigens. The variants associated with the C antigen were: 7 r?s, 4 RHD(1-2)-CE(3-9)-D(10), 1 RHD(M275I) and 1 new null allele dubbed RHCE(1-2)-RHD(3361del11-10) whereas those associated with the E antigen were: 5 RHD(46T>C) and 1 new null allele named RHD(581insG)responsible for a premature stop codon at position 197. The 5 samples carrying the RHD(46T>C) allele were further characterized serologically using an extended set of anti-D clones and absorption-elution assays. The results confirmed an extremely weak expression of the D antigen in all samples carrying this mutation. Moreover, in two of these samples an altered E antigen expression was also found associated with the RHcE(697C>G, 712A>G, 733C>G, 744T>C) allele. Conclusions: Molecular analyses showed that the RHD deletion is the main cause (97.9%) of the D negative phenotype in the population studied. However, a 2.1% of the samples analysed carried DEL or null variants with this finding being more frequent in D negative samples expressing C and/or E antigens, as previously reported. D negative/RHD positive samples were indeed found in up to 16.7% in this latter group. RHDØ and r?s reveal the contribution of the African ethnicity to the genetic pool. Interestingly, two new mutations were found in the admixed population analyzed, one of them being a hybrid structure between a previously described RHD null allele recombined with RHCE sequences. The understanding of the genetic background of the RH locus in our population will help to develop reliable strategies in Blood Banks and prenatal RHD genotyping.