Molecular analysis of two RH allelic variants

MUFARREGE N; TRUCCO BOGGIONE C; PRINCIPI C; BRAJOVICH, MELINA ELIANA LUJÁN; MATTALONI S; ENSINCK A; GARCÍA BORRÁS S; BIONDI C; COTORRUELO C

Rosario

Congreso; XX Congreso y XXXVIII Reunión Anual de la Sociedad de Biología de Rosario; 2018

The Rh system is one of the most important blood groups in transfusion medicine since its antibodies are involved in hemolytic transfusion reactions. The RH locus is formed by the RHD and RHCE genes that encode homonymous proteins located in the erythrocyte membrane. Rh polypeptides express more than 50 antigens, of which D epitope is the most immunogenic. The D variant phenotype (Dvar) is characterized by a decreased expression of the D antigen in the red blood cell membrane. Molecular biology studies are fundamental for understanding the genetic complexity responsible for these variants. The aim of this work was to characterize the molecular bases of two Dvar phenotype samples of donors who attended the Immunohematology Laboratory of the Biochemical and Pharmaceutical Sciences Faculty, National University of Rosario. Rh phenotype was determined by hemagglutination techniques using IgM and IgG monoclonal antibodies (clones TH28 / MS26), IgM (clone MS201), IgM (clone RUM1) and IgM (clones LDM1 and ESD1M) for the study of the D antigen. Monoclonal antibodies anti-C (clone MS24), anti-c (clone MS33), anti-E (clone MS258 / MS80) and anti-e (clones MS16 + MS21 + MS63) were used for C, c, E and e antigens expression analysis, respectively. Genomic DNA of these samples was obtained through the salting-out method. RHD zygosity was studied by PCR-RFLP. Weak allelic variants D type 1, 2, 3 and 4 was analyzed using specific PCR for these alleles. Subsequently, the characteristic polymorphisms of each of the RHD 10 exons were investigated by PCR-SSP. Finally, sequencing analysis was carried out by Next Generation Sequencing (NGS). Both samples showed a phenotype Dvar, C +, c +, E-, e +. D antigen study showed weak reactions in immediate reading that were enhanced in antiglobulin phase. A hybrid Rhesus box was detected in each sample, indicating a hemizygous RHD state. Molecular strategies did not show the most frequent D variant alleles presence. NGS analysis showed the presence of the 851C> T point mutation in RHD exon 6 in one of the samples, while in the other the 881C> T polymorphism was detected in RHD exon 6. The detected SNPs are responsible for the amino acid substitutions Ser284Leu and Ala294Val, respectively and are located in the transmembrane region of the RhD protein. Both aminoacidic changes -a polar amino acid (AA) by a hydrophobic one (Leu) in one case, and a small hydrophobic AA (Ala) by a larger one (Val) in the other one- would lead to an incorrect folding of the RhD polypeptide, which would result in a decrease in the expression of D epitopes. In transfusions, the identification of alleles that originate phenotypes with aberrant expression of the RhD protein contributes to hemocompatibility based unit selection and to clinical decision making.