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

Abstract

The molecular basis for RHD pseudogene or RHDΨ is a 37-bp insertion in exon 4 of RHD. This insertion, found in two-thirds of Dnegative Africans, appears to introduce a stop codon at position 210. The hybrid RHD-CE-D s , 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 RHDΨ and RHD-CE-D s . DNA from 206 blood donors were tested for RHDΨ by a multiplex PCR that detects RHD, RHDΨ 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%) of the 206 D-negative Brazilians studied had the RHDΨ, 5 (2%) had the RHD-CE-D s hybrid gene associated with the VS+V-phenotype, and 179 (87%) 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%) had inactive RHD and 3% had the RHD-CE-D s 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.

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“…It was also surprising to find DAU-0 as the most prevalent aberrant RHD allele in Mali, an allele which was previously perceived as rare and occurring in Europeans [9]. In contrast, the frequencies of RHDΨ and Ccde s in Mali were similar to previous reports [5,13,14] for other African populations.…”

“…Clinical experience and screens for certain RHD alleles hinted to a high incidence of aberrant RHD in Africans [5,6,13], but a comprehensive investigation of the RHD allele distribution was lacking for any African population. Such systematic knowledge could have considerable impact for typing and transfusion strategy in populations with African admixture; possible difficulties in transfusion therapy and in genotyping could be anticipated and appropriately improved strategies devised.…”

Noninvasive fetal RHD genotyping from maternal plasma in an admixed Brazilian population

“…This percentage is higher than that reported in studies performed in the UK, China, and the USA, which varied from 3.4 to 6% (Bianchi and Lo, 2001;Finning et al, 2008). This is probably due to the heterogeneous genetic profile of the Brazilian population, which raises the possibility that different variants, rare in other populations, prevent the amplification of some exons by mismatching of primers and/or probes with their targeted genomic regions (Rodrigues et al, 2002). Therefore, in ad-mixed populations in which a high variety of RHD alleles is expected, fetal RHD genotyping may lead to false-negative results and may hinder the proper management of RhD-negative pregnant women.…”

Detection of fetal RHD pseudogene (RHDΨ) and hybrid RHD-CE-Ds from RHD-negative pregnant women with a free DNA fetal kit

“…In contrast, 82% of D-negative black Africans do not have a homozygous deletion of RHD, but carry one or two RHD variant genes, the RHD pseudogene (Singleton et al, 2000) or the RHD-CE-D s hybrid gene (Faas et al, 1997;Rodrigues et al, 2002). In one of the largest validation studies published on non-invasive fetal RHD genotyping, Rouillac-Le et al (2004) amplified RHD exon 7 and exon 10 in 893 maternal plasma samples.…”