A DFR SEROLOGICAL PATTERN CAUSED BY A NOVEL MISSENSE MUTATION

MUFARREGE, NICOLÁS; TRUCCO BOGGIONE, CAROLINA; PUPPO, M; ABATEMARCO, V; IZAGUIRRE, S; LUJÁN BRAJOVICH, MELINA ELIANA; MATTALONI, STELLA MARIS; BIONDI, CLAUDIA; KUPERMAN, S; COTORRUELO, CARLOS

Copenhague

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 transfusioncompatibility and in obstetrics to define strategies for medical intervention in pregnant women. In most cases, serologicaldeterminations are inconclusive and in this context the study at DNA level is appropriate to overcome limitations.Aims: the aim of this study was to characterize the molecular background of two D variant (Dvar) samples referred to our laboratoryfor RHD genotyping.Methods: commercially available monoclonal reagents were used to evaluate the Rh phenotype (D, C, c, E, e) of donors? red bloodcells through standard tube hemagglutination techniques including the indirect antiglobulin test (IAT). Particularly, D antigen statuswas 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), and 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. RHD zygosity status was analysed by aPCR-RFLP method. Weak D types were evaluated by allele-specific PCRs. Samples were subjected to a RHD exon scanningprocedure based on PCR-SSP to analyse multiple RHD exon polymorphisms. Direct automated sequencing on PCR products of the10 RHD exons was also performed.Results: direct tube testing showed a weak reactivity with all anti-D reagents used. IAT enhanced the blended anti-Dshemagglutination. The Rh phenotype was Dvar, C+, c-, E-, e+ for proband #1 and Dvar, C+, c+, E-, e+ for proband #2. Both samplesshowed a clear DFR reaction pattern when tested with the monoclonal anti-D reagents of the ID-Partial RhD Typing Set. One hybridRhesus box was detected in each sample suggesting a hemizygous status for the RHD locus. Neither allele-specific PCRs nor exonscanninganalyses showed modifications in the polymorphisms studied. Interestingly, genomic DNA sequencing revealed a new RHDvariant characterized by the point mutation 325A>G in RHD exon 2.Summary / Conclusions: we report a novel RHD allele with a missense mutation responsible for the aminoacidic changeThr109Ala, predicted to be in the extracellular boundary of the fourth transmembrane segment of the RhD protein. The ID-PartialRhD Typing Set indicated a DFR phenotype in both samples. However, DNA sequencing showed a different genetic backgroundfrom the DFR-1, DFR-2, DFR-3, DFR-4 and DFR-5 alleles described so far. While these variants result from hybrid structuresinvolving RHD exon 4 (and exon 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. Our findingsshow that molecular studies are a necessary complement for characterizing samples with variant D phenotypes. RHD genotyping maycontribute for clinical decision making in transfusion and to provide appropriate recommendations for anti-D prophylaxis inpregnancies.