A DFR serological pattern caused by a novel missense mutation

MUFARREGE N; TRUCCO BOGGIONE C; PUPPO M; ABATEMARCO V; IZAGUIRRE S; LUJÁN BRAJOVICH M; MATTALONI S; BIONDI C; KUPERMAN S; COTORRUELO C

Copenague

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

International Society of Blood Transfusion

Background: the identification of aberrant expressions of the D antigen is critical in blood banks to optimize transfusion compatibility and to ensure safe blood provision and, in Obstetrics to define strategies for medical intervention in pregnant women. In most cases, serological determinations are inconclusive and in this context the study at DNA level is appropriate to overcome these limitations.Aim: the aim of this study was to characterize the molecular background of two D variant (Dvar) samples referred to our laboratory for RHD genotyping.Methods: commercially available monoclonal reagents were used to evaluate the complete Rh phenotype of donors? red blood cells (RBCs) through standard tube hemagglutination techniques including the indirect antiglobulin test (IAT). The D antigen status was examined with two blended IgM/IgG anti-Ds (clones TH-28 + MS-26; Wiener Lab, Rosario, Argentina and clones P3x61 + P3x21223b10 + P3x290 + P3x35; Diagast, Loos, France), an three IgM anti-Ds (clone MS-201, clone RUM-1 and clones LDM1 + ESD1M; Rediar, Buenos Aires, Argentina). Partial D phenotypes were studied using the ID-Partial RhD Typing Set (DiaMed, Cressier, Switzerland). A modified salting-out method was used to isolate genomic DNA. Weak D types were evaluated by allele-specific PCRs. Exon-scanning and sequencing analyses of the 10 RHD exons were also performed. Results: direct tube testing showed a weak reactivity with all anti-D reagents used. IAT enhanced the blended anti-Ds hemagglutination. The complete Rh phenotype was Dvar, C+, c+, E-, e+ for each proband. Both samples showed a DFR reaction pattern when tested with the monoclonal anti-D reagents of the ID-Partial RhD Typing Set. Neither allele-specific PCRs nor exon-scanning analyses showed modifications in the polymorphisms studied. Interestingly, genomic DNA sequencing revealed a novel allele characterized by the point mutation 325A>G in RHD exon 2.Conclusions: we report a novel RHD allele with a missense mutation that is responsible for the aminoacidic change Thr109Ala, predicted to be in the extracellular boundary of the fourth transmembrane segment of the RhD protein. The ID-Partial RhD Typing Set indicated a DFR phenotype. However, DNA sequencing showed a different genetic background from those DFR variants described so far. While DFR-1, DFR-2, DFR-3, DFR-4 and DFR-5 result from hybrid alleles involving RHD exon 4 (and 3 in DFR-5), a point mutation in RHD exon 2 is responsible for the variants reported in this work. Serologic and molecular results suggest a genetic association in cis between this new RHD variant and the RHCe allele. The reduced expression of the D antigen may be caused by alterations during the polypeptide assembly to the RBC membrane. Our findings show that molecular studies are a necessary complement for characterizing the samples with variant D phenotypes. RHD genotyping may contribute for clinical decision making in transfusion and to provide appropriate recommendations for anti-D prophylaxis in pregnancies.