Presence of the RHD pseudogene and the hybrid RHD-CE-Ds gene in Brazilians with the D-negative phenotype

RESUMO

The molecular basis for RHD pseudogene or RHDpsi is a 37-bp insertion in exon 4 of RHD. This insertion, found in two-thirds of D-negative Africans, appears to introduce a stop codon at position 210. The hybrid RHD-CE-Ds, where the 3' end of exon 3 and exons 4 to 8 are derived from RHCE, is associated with the VS+V- phenotype, and leads to a D-negative phenotype in people of African origin. We determined whether Brazilian blood donors of heterogeneous ethnic origin had RHDpsi and RHD-CE-Ds. DNA from 206 blood donors were tested for RHDpsi by a multiplex PCR that detects RHD, RHDpsi and the C and c alleles of RHCE. The RHD genotype was determined by comparison of size of amplified products associated with the RHD gene in both intron 4 and exon 10/3'-UTR. VS was determined by amplification of exon 5 of RHCE, and sequencing of PCR products was used to analyze C733G (Leu245Val). Twenty-two (11 percent) of the 206 D-negative Brazilians studied had the RHDpsi, 5 (2 percent) had the RHD-CE-Ds hybrid gene associated with the VS+V- phenotype, and 179 (87 percent) entirely lacked RHD. As expected, RHD was deleted in all the 50 individuals of Caucasian descent. Among the 156 individuals of African descent, 22 (14 percent) had inactive RHD and 3 percent had the RHD-CE-Ds hybrid gene. These data confirm that the inclusion of two different multiplex PCR for RHD is essential to test the D-negative Brazilian population in order to avoid false-positive typing of polytransfused patients and fetuses