Molecular characterization of D negative C positive samples harboring the RHD-CE(3-9)-D hybrid allele

MUFARREGE, NICOLÁS; TRUCCO BOGGIONE, CAROLINA; PRINCIPI, CINTIA; LUJÁN BRAJOVICH, MELINA ELIANA; MATTALONI, STELLA MARIS; ENSINCK, MARÍA ALEJANDRA; GARCIA BORRAS, SILVIA; BIONDI, CLAUDIA; COTORRUELO, CARLOS

Rosario

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

Sociedad de Biología de Rosario

The RHD locus is highly polymorphic and has a large number of alleles responsible for different D phenotypes. RHCE and RHD genes reside in close proximity, share high nucleotide homology, and harbor numerous repetitive elements, which may serve as hot spots for recombination, giving rise to new alleles to be discovered. Silent RHD alleles generate a negative D phenotype, while DEL variants produce apparent D negative phenotypes that express a minimal amount of D antigen in the erythrocyte membrane detected only by specialized serological techniques. In addition, numerous RHD alleles are responsible for a variant D phenotype. Previous research conducted in our laboratory showed a high incidence of the DVII variant allele in our population responsible for a weakened expression of the D antigen. On the other hand, RHD exon 2 sequencing analysis performed in four D negative samples carrying the RHD-CE(3-9)-D hybrid structure detected the 329T>C mutation, characteristic of the partial DVII allele, in two of the samples. The aim of this study was to characterize the molecular background of D negative C positive samples harboring the RHD-CE(3-9)-D hybrid allele. 681 D negative, C positive samples were selected for this study. The D, C, c, E and e status was determined by standard serologic hemagglutination techniques using specific monoclonal antibodies. In particular, the D antigen was evaluated with a blended anti-D. When an immediate spin negative result was observed with the latter antiserum, the samples were tested by the indirect antiglobulin test. Genomic DNA was isolated using a modified salting-out method. DNA samples were initially screened for the presence of intron 4 and the 3? untranslated region of the RHD gene using PCR strategies. RHD zygosity was studied by PCR-RFLP in those D negative samples carrying RHD specific sequences. In addition, these D negative RHD positive samples were further characterized by RHD exon specific PCRs. Finally, samples lacking RHD exons 3 to 9 were analysed to detect the 329 T>C mutation responsible for the partial DVII allele through a PCR-SSP strategy. Among the 681 D negative samples expressing C antigen, 115 (16.9%) showed RHD specific amplifications. One hybrid Rhesus box was detected in all D negative RHD positive samples suggesting a hemizygous status. The RHD-CE(3-9)-D hybrid allele was found in 18 (15.7%) of the 115 samples carrying RHD specific fragments. Surprisingly, the 329 T>C mutation was detected in 8 (44.4%) of the 18 samples harboring the RHD-CE(3-9)-D hybrid allele. In this work a new silent allele, dubbed RHD(329T>C)-CE(3-9)-D, was found to have a high incidence among the RHD-CE(3-9)-D hybrid structure. Interestingly, the detection through PCR-SSP of the 329T>C mutation, characteristic of the partial DVII allele, in eight samples, supports our previous suggestion that a homologous recombination event between a DVII allele and RHCE sequences could be responsible for this new hybrid structure. The accurate detection of the RHD(329T>C)-CE(3-9)-D hybrid allele in RHD genotyping strategies in our population would prevent misleading results when serological data are not available.