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

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-Ds, where the 3' end of exon 3 and exons 4 to 8 are derived from RHCE, is associated with the VS+Vphenotype, 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-Ds. 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-Ds hybrid gene associated with the VS+Vphenotype, 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 RHDCE-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. Correspondence


Introduction
The Rh blood group system is clinically important because it is involved in hemolytic disease of the newborn, hemolytic transfusion reactions and autoimmune hemolytic anemia.Rh is a highly complex red cell blood group system with 46 antigens (1,2).
The most important antigens are D, C/c, and E/e.The Rh system antigens are encoded by two homologous genes (3), the RHD gene and the RHCE gene, both located on chromosome 1p34.3-p36.1 (4).RHCE gives rise to C/c and E/e polymorphism and RHD encodes the RhD antigen (5).
Total or partial deletion of the RHD gene can result in the D-negative phenotype (3,(6)(7)(8)(9).In non-Whites, D-negativity can appear in individuals carrying the complete RHD gene (10,11).This group includes individuals of black or Asian origin (10,11) who exhibit either an internal duplication (12) or a deletion (13) within the RHD gene, resulting in a premature stop codon in RHD transcripts.The presence of certain RHD regions in hybrid genes encoding partial D antigens may predict a D-negative phenotype, and the presence of some RHD regions in genes encoding no D antigen may predict a Dpositive phenotype.In order to avoid these complications, methods which detect more than one region of RHD have been introduced (11,14,15).About two-thirds of D-negative Africans have an inactive RHD gene (12).This pseudogene (RHDΨ) has a 37-bp insert in exon 4, which may introduce a reading frame shift and premature termination of translation and a translation stop codon in exon 6 (12).Of the remaining one-third of African D-negative donors, about half appear to be homozygous for an RHD deletion and about half have the RHD-CE-D s hybrid gene characteristic of the (C)ce s haplotype that produces c, VS, and abnormal C and E, but not D (8,12).In D-negative African Americans and South African people of mixed race, the same three genetic backgrounds are present, but 24% of African Americans and 17% of South African donors of mixed race have RHDΨ, and 54% of African Americans and 81% of South African donors of mixed race have no RHD (12).
In the present study we investigated whether D-negative Brazilian blood donors of heterogeneous ethnic origin had altered RHD.We studied DNA samples from 206 D-negative blood donors (50 of Caucasian descent and 156 of African descent) by two different multiplex PCR that detect RHD, D variants, RHC/c and the RHDΨ and by sequencing exon 5 of RHCE for the 733 C>G polymorphism (VS antigen).Our observa-tion was in agreement with previous publications showing that RHD was deleted in all individuals of Caucasian descent.However, 14% of D-negative Brazilians of African descent studied had the RHDΨ and 3% had the RHD-CE-D s hybrid gene.These data show the necessity of performing multiplex PCR for detecting more than one region of RHD and the 37-bp insertion in populations of African descent for predicting the D phenotype from DNA in order to avoid falsepositive typing of polytransfused patients and fetuses.

Blood donors
We studied peripheral blood samples from 206 random D-negative blood donors (50 of Caucasian descent and 156 of African descent) who agreed to participate in this study by signing an informed consent form.The study was approved by the Medical Ethics Committee of UNICAMP and CONEP.

Agglutination tests
RhD phenotypes were determined by hemagglutination in gel cards (Diamed AG, Morat, Switzerland) using two different commercial sources of monoclonal antisera (Gamma Biologicals Inc., Houston, TX, USA; Diamed AG).VS and V phenotypes were determined by standard techniques using polyclonal antibodies (patient serum).

DNA preparation
DNA was extracted from blood samples using the DNAzol (Gibco BRL, Rockville, MD, USA) and a blood DNA purification kit (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA) according to manufacturer recommendations.
Allele-specific PCR for RHD genotyping-PCR analysis for the presence of RHD was performed in two genomic regions, intron 4 and exon 10.Briefly, PCR was performed with 100-200 ng of DNA, 50 pmol of each primer, 2 nmol of each dNTP, 1.0 U Taq DNA polymerase, and buffer in a final volume of 50 µl.PCR was carried out in a thermal cycler (9700, Perkin Elmer, Foster City, CA, USA) and the same profile was used for both assays, as follows: 15 min at 95ºC, 35 cycles of 40 s at 94ºC, 40 s at 62ºC, and 1 min at 72ºC, followed by 10 min at 72ºC.Amplified products were analyzed by electrophoresis in 1.5% agarose gel in Trisacetate EDTA buffer.For exon 10, a common 5' primer (EX10F) was used for both RHD and RHCE.When paired with the RHDspecific 3'-untranslated region (UTR) primer, it produced a product of 210 bp, and when paired with the RHCE-specific 3'-UTR primer, a product of 163 bp ( 16) was produced.A set of three primers, RHI41 and RHI42 (previously reported; 16), and an additional third primer RHI43 were used for intron 4. The combination of these three primers generates products of 115 bp for RHD and 236 bp for RHCE (Figure 1).The sequences of the primers are listed in Table 1.

Multiplex PCR for the presence of the RHD pseudogene
Analysis of the RHDΨ 37-bp insert was performed using a multiplex PCR that detects the presence of D, differentiates RHC/c and identifies RHDΨ (12).PCR primers are listed in Table 2. Thirty cycles of PCR were performed at 94ºC for 1 min, 65ºC for 1 min, and 72ºC for 3 min 30 s. PCR products were analyzed by 2% agarose gel electrophoresis (Figure 2B).

Multiplex PCR for RHD variants
Analysis of RHD variants was performed in all samples using an RHD multiplex assay directed at six regions of RHD (exons 3-7 and exon 9), covering all exons with RHDspecific sequences in the coding regions (15).The multiplex PCR was performed in a thermal cycler (9700, Perkin Elmer) with the following cycle specifications: 32 cycles of 1 min at 95ºC, 1 min at 55ºC and 45 s at 72ºC, followed by 10 min at 72ºC.PCR products were size-separated by 8% acrylamide gel electrophoresis (Figure 2A).PCR primers are listed in Table 3.

Sequence analysis
Sequence analysis was performed on PCR products amplified from genomic DNA using RHCE-specific primers for exon 5 (Table 4) to determine the presence of 733G predicted to encode Val245 (VS+) and RHDspecific primers for exon 3 (Table 4) to determine the presence of the D-CE hybrid.PCR products were purified on 1% agarose gels using a Qiaex II gel extraction kit (Qiagen, Valencia, CA, USA), and sequenced directly using an ABI 373XL Perkin Elmer Biosystems sequencer.alloanti-D reagents that react with all known partial D and weak D antigens.Red blood cells from five black donors who were Dnegative were phenotyped as C+c+E-e+ VS+ and V-.These five donors all showed a weak expression of C.

Screening D-negative donors for exon 10 and intron 4
All D-negative donors were tested by the allele-specific PCR method designed to determine the presence of RHD exon 10 and intron 4 (Figure 1).Three patterns of reaction were apparent: presence of both RHD regions, absence of both RHD regions, and presence of RHD exon 10, but absence of RHD intron 4. Of the 206 D-negative Brazilian blood donors tested, 87% lacked RHD (50 of Caucasian descent and 129 of African descent), 11% had both regions of RHD, and 2% had only RHD exon 10 (Table 5).

Screening D-negative donors for RHD exons 3-7 and exon 9
Multiplex PCR to detect D variants (Figure 2A) in selected donors with RHD revealed that donors with RHD exon 10 and intron 4 also had RHD exons 3, 4, 5, 6, 7, and 9, suggesting the presence of a grossly intact RHD.Red cells from five donors of African descent with RHD exon 10, but without RHD intron 4, were C+ and VS+V-.In addition to RHD exon 10, donors of this type had RHD exon 9 and a hybrid exon 3 comprising a 5' end derived from RHD and a 3' end derived from RHCE.The presence of the 773G mutation in exon 5 of the RHCE determined by sequencing confirmed the VS antigenicity.

Serology
Red blood cells from the 206 blood donors gave D-negative results with two  reported among Caucasians with the less frequent Ce and cE haplotypes and among D-negative individuals of African descent (10,14,17,18).The RHDΨ, characterized by an insertion of 37 bp leading to a premature stop codon, can inadvertently cause discrepancy in genotype/phenotype correlation unless a specific assay (12) for detecting this insertion is employed.RHDΨ is found in Dnegative South Africans (66%) and in African Americans (24%) (12).In our study, 11% of the 206 D-negative Brazilians studied had this nonfunctional RHD.
An RHD-CE-D fusion gene, in which the 3' end of exon 3 plus exons 4-8 is derived from RHCE, is sometimes associated with a D-negative phenotype in people of African This suggests that these five donors (2%) have the RHD-CE-D gene associated with the (C)ce s complex (RHD-CE-D s ) (Table 5).Donors with neither exon 10 nor intron 4 of RHD also lacked RHD exons 3, 4, 5, 6, 7 and 9.

Genomic DNA analysis by sequencing
Genomic DNA analysis performed by sequencing revealed in five donors of African descent the presence of the D-CE hybrid exon 3 and the 733G mutation [predicted to encode Val245 (VS+)], associated with the RHD-CE-D s hybrid gene (Table 5).

Discussion
There are actually three genetic mechanisms associated with the D-negative phenotype: deletion of RHD (3), an RHD pseudogene containing a 37-bp insert and one or two stop codons (12), and a hybrid RHD-CE-D s gene that probably produces an abnormal C antigen but does not produce a D antigen (8,12).RHD is generally absent in RHD-negative Caucasians carrying the cde haplotype.However, exceptions have been origin (8,12).The hybrid gene carries a Leu245Val substitution responsible for the VS antigen and is associated with the presence of a weak C. We found this hybrid gene in five donors of African descent phenotyped as D-C weak c+E-e+VS+V-.These samples were D-positive by exon 10 analysis but Dnegative by intron 4 and exon 7 analysis.The five samples were all heterozygous for C733G in exon 5 of RHCE which predicts a Leu245Val (VS antigen) and so had a probable ce/(C)ce s genotype.These findings, taken together with a previous report that RHDΨ is of high prevalence in populations of similar background (10), strongly suggest that genotype determination of RH must include a thorough analysis of RHD.In the present study, we used two multiplex PCR, one (15) to detect gross chromosomal alterations in RHD and RHCE including gene rearrangement and hybrid genes, and the other (12) to detect RHDΨ.Furthermore, the multiplex PCR that detects RHDΨ has the advantage of determining C/c at the DNA level in the presence of RHD, a feature that is desired in transfusion practice and to predict the RhD blood type of a fetus in populations of African descent.Typing the fetus for the RHC allele is also valuable because anti-G may be responsible for hemolytic disease of the newborn.
The most common D-negative Rh haplotype in Africans is RHDΨ with the ce allele of RHCE.The 37-bp insert in exon 4 of RHDΨ is a duplication of a sequence spanning the boundary of intron 3 and exon 4.This insert may introduce a reading frame shift and a translation stop codon at position 210.However, the duplication introduces another potential splice site at the 3' end of the inserted intronic sequence in exon 4 and another stop codon in exon 6 of the gene (12).RHD mRNA was not detected in Dnegative individuals with RHDΨ, despite the presence of RHCE transcripts.In fact, Africans with RHDΨ are truly D-negative since they can produce anti-D and cause hemolytic disease of the newborn as previously reported (12).
Our results confirm the necessity to perform multiplex PCR including gene rearrangement and hybrid genes and the RHDΨ in populations of African descent for the appropriate management of transfused patients and for RhD-negative pregnant women who are sensitized, particularly when the fetal RHD is determined by molecular assays.
Finally, the 11% prevalence of RHDΨ suggests a high degree of admixture of individuals of African descent in the Brazilian population.

Figure 1 .
Figure 1.RHD genotyping by intron 4 and exon 10 of RHD.Lanes 1 and 3: RHD-positive samples display bands of 236 bp for RHCE and 115 bp for the RHD intron 4 sequence.Lane 2: RHD-negative sample displays only the 236-bp band corresponding to the RHCE intron 4 sequence.Lane 4: Reaction blank.Lane 5: 50-bp DNA ladder.Lanes 6 and 8: RHD-positive samples that amplify a 245-bp product of the RHD and a 160-bp product of the RHCE exon 10 sequence.Lane 7: RHD-negative sample displays only the 160-bp band corresponding to the RHCE exon 10 sequence.

Figure 2 .
Figure 2. Multiplex PCR for detection of the RHD hybrid alleles and the RHDΨ.A, 8% acrylamide gel showing results with multiplex PCR products amplified with six RHD-specific primer sets (exons 3, 4, 5, 6, 7 and 9).B, 2% agarose gel showing results with multiplex PCR for predicting D and C/c phenotype and for detecting the presence of RHDΨ.

Table 1 .
Primers used for RHD genotyping.

Table 3 .
Primers used for identification of the RHDΨ 37-bp insert.

Table 4 .
Primers used for sequence analysis of VS and D-CE hybrid.

Table 5 .
Results of testing for the presence or absence of RHD, RHDΨ and RHD-CE-D s in 206 Brazilian blood donors.