Oral Presentation: Molecular study of D negative and D variant phenotypes in Argentine

GARCÍA BORRÁS S; COTORRUELO C; DORIGAN DE MACEDO M; MATTALONI S; MUFARREGE N; CASTILHO L; LUJÁN BRAJOVICH M; TRUCCO BOGGIONE C

Copenague

Congreso; 27th Regional Congress of the International Society of Blood Transfusion; 2017

International Society of Blood Transfusion

Background: the current population of Argentine is the result of generations of intermixing between Amerindians, Europeans and Africans. The contribution of these ethnic groups to the genetic pool varies in different areas of the country. In these sense, a comprehensive study of the RH locus in our population is needed.Aim: the aim of this study was to investigate the RHD molecular polymorphism in different geographical regions of Argentine.Materials and Methods: 796 D negative, C/E positive and 324 variant D samples from North and Central areas of the country were studied. The D, C, c, E and e status was determined by standard serologic hemagglutination techniques. DNA samples from D negative, C/E positive individuals were initially screened for the presence of intron 4 and the 3? untranslated region of the RHD gene using PCR strategies. RHD zygosity was investigated by PCR-RFLP in those D negative samples carrying RHD specific sequences and in all variant D phenotypes. Allele characterization was performed by PCRs, microarray and sequencing.Results: in the 796 D negative C/E positive phenotypes, RHD specific amplifications were detected in 133 samples (16.71%). Silent RHD alleles were identified in 12.19%, DEL alleles in 2.51% and no molecular polymorphisms were found in 2.01% of the samples. Among the 133 D-/RHD+ samples, the following silent alleles were characterized: RHD-C-Ds (43.61%), RHD*581insG (14.29%), RHD-CE(2-9)-D (6.02%), RHD(329T>C)-CE(3-9)-D (5.26%), RHD-CE(4-8)-D (0.75%), RHD-CE(4-7)-D2 (0.75%), RHD(1-2)-RHD(3361_371del11-10) (0.75%), RHDψ (0.75%) and the novel RHD*1001A (0.75%). DEL alleles were: RHD*46C (9.03%), M295I (3.01%), RHD(IVS3+1g>a) (1.50%), RHD-CE(4-9)-D (0.75%), RHD*1248insG (0.75%).In the group of the 324 variant D phenotypes, the 58.02% carried a weak D type 1, 2 or 3 allele. In the rest of the samples, the following variants were found: weak D type 4 (14.81%), RHD*DVI (4.63%), RHD*DVII (1.54%), DFR-2 (0.92%), weak D type 59 (0.62%), weak D type 5 (0.62%), RHD*DMH (0.62%), weak D type 15 (0.31%), weak D type 45 (0.31%), weak D type 48 (0.31%), RHD*DIV type 5 (0.31%), RHD*DVa (0.31%), weak D type 1/RHD-CE-Ds (0.31%). Also 5 novel RHD alleles were characterized: weak D type 93 (9.57%), RHD*325G (0.31%), RHD*763A (0.31%), RHD*764A (0.31%) and RHD*911A (0.31%). Weak D type 93 was identified only associated to ccEe phenotype. No molecular polymorphisms were found in 5.55% of the variant D samples analyzed. It is worth mentioning that the distribution of samples carrying different RHD alleles varied significantly between North and Central areas of Argentine.Conclusions: the results obtained allowed a comprehensive analysis of the RHD locus polymorphism in our country. To note, weak D type 93, RHD*46C and RHD*581insG alleles were most frequently found in samples of individuals from North Argentine and have not been previously reported in other populations. Considering that the Amerindian influence is greater in the North region, these RHD variants could be associated to Native populations. Our findings show the relevance of RHD genotyping for a better management of D negative units in Blood Banks and prenatal immunoprophylaxis. Further studies are being performed in samples with no molecular polymorphisms found.