The Rhesus (Rh) blood group system contains antigens produced from two distinct genes, RHD and RHCE, that are tandemly localized on chromosome 1 and are thought to have arisen through duplication of a single ancestral gene (15,16). The RH genes are greater than 95% homologous at the nucleotide sequence level, both consist of 10 exons spanning over 75 kb, and both encode peptides of 417-amino-acid residues, with a predicted molecular mass of 30-35 kDa
(17-19). Approximately 15% of Caucasians are RhD-negative, where the D-negative phenotype is the result, in general, of absence of the RHD gene; such individuals, however, possess two copies of the RHCE gene. There are a number of sequence differences between the RHCE and RHD genes, which can be utilized in genotyping assays to identify the presence or absence of the RHD gene as well as detect the RhC/c- and RhE/e-specific polymorphisms within the RHCE gene. The Rh antigens are among the most antigenic of the polymorphic red cell surface proteins making them highly clinically significant, especially in the context of HDN (20). HDN resulting from RhD has become relatively infrequent because of the use of Rh immune globulin (RhoGAM), given to RhD-negative mothers giving birth to D-positive infants, thereby passively eliminating circulating and potentially immunogenic fetal red cells before they are recognized by the maternal immune system (21).
2.1. GENOTYPING THE RHD RED CELL ANTIGEN SYSTEM Polymerase chain reaction (PCR)-based RhD typing assays, utilizing sequence differences in intron 4, exon 3, exon 7, and the untranslated region within exon 10, have been previously described (18,22-25). However, discrepancies between serotyping and genotyping have been observed. These discrepancies are largely the result of the existence of allelic variants, the molecular basis of which is often the result of recombination between the RHD and RHCE genes. In these hybrid genes, some RHD sequences are replaced with the corresponding sequences from the RHCE gene (22-26). For other variants, all that is known is that the sequences targeted by PCR primers or restriction enzymes are altered or deleted. RhD genotyping is commonly accomplished by multiplexing the oligonucleotide primers of Arce et al. with those described by Bennett et al (18,23). The RhD genotyping strategy described by Arce et al., detects a 600-bp deletion within intron 4 of the RHD gene, which is not present within intron 4 of the RHCE gene (18). Amplification with a primer pair that targets exons 4 and 5 of the RhD and the RhCcEe genes results in a 1200-bp RhCcEe product and a 600-bp product in RhD-positive individuals. The RhD-specific oligonucleotide primers described by Bennett et al. specifically amplify a 193-bp product of the 3' untranslated region of exon 10 (18,23). RhD genotyping can also be performed through specific amplification of a 96-bp product from exon 7 of the RHD gene, previously described by Simsek et al. (25). Finally, specific amplification of a 111-bp product from exon 3 of the RHD gene can be used for genotyp-ing, as previously described by Beckers et al. (22).
Among Caucasians, a high concordance between serology and genotyping has been observed when methods targeting sequence differences in intron 4 and the untranslated region within exon 10 are combined (Fig. 1); however, false-positive results, especially among African and Asian individuals, have been observed (22-25). Less than 5% of Native South Africans are serologically RhD-negative; however, less than 20% of serologically D-negative individuals have been observed to completely lack the RHD gene (27). In 66% of serologically D-negative native Africans, a silent RHD gene (RHDy) has been identified that possesses a 37-bp insertion in exon 4, which presumably produces a premature stop codon at position 210, resulting in a truncated protein (27). Discordance between RhD serology and RHD genotyping results has been a potential problem for laboratories conducting prenatal genotyping (28-30). Therefore, we evaluated and augmented our standard RHD genotyping assay, which specifically targets exon 10 of RHD and the intron 4 600-bp deletion/insertion polymorphism of the RHD/RHCE genes, with an additional reaction possessing primers that identify the 37-bp exon 4 insertion that gives rise to the RHD^allele (27). Primers flanking the exon 4 insertion point were used for detection of RHD and RHDy among a total of 231 serotyped individuals; 134 African-American, 85 Caucasian, and 12 RhD serotype-negative/genotype-positive, D-sensitized women (Fig. 1) (14). RHDy was detected in 19% (n = 4/21) of RhD sero-negative African Americans and 4.4% (n = 5/113) of RhD sero-positive African-Americans (14). Complete concordance was observed, with this additional primer set, between serology and genotyping when detecting the 381-bp normal RHD PCR product, whereas detection of the 418-bp RHD^gene product was useful in resolving 10/12 previously ambiguous prenatal genotypings where the RhD-sensitized mother possessed an apparently intact RHD gene in the standard assay (14). In these cases, it was possible, with the RHD^primer set, to determine the basis for the discrepancy between the maternal serotype and genotype and, second, to determine if the fetus had paternally inherited RHD or maternally inherited RHDy, or both. The addition of this primer set to RHD genotyping strategies enables definitive genotyping of most RhD-negative African-Americans based on the reported frequencies of 54% and 24% for the homozygous deletion of RHD and RHD (//alleles, respectively, as the genetic basis for D-negativity (27). In our study, 19% (n = 4/21) of the RhD sero-negative African-American donors possessed RHDy, which is consistent with previous reports (14,27).
Fifty-six percent of the RhD-positive individuals within the Caucasian population are heterozygous for RhD (31), making knowledge of the father's zygosity useful for predicting the probability of a couple's conception of an RhD-positive pregnancy, especially if the mother has been immunized through a previous pregnancy. Recently, reliable approaches utilizing real-time quantitative PCR and ASPCR have been reported that allow determination of RHD gene dosage (32). These assays can reduce the need for amniocentesis, because a homozygous RHD-positive male can only produce RhD-positive offspring, making the invasive procedure unnecessary. However, heterozygous males have a 50% chance of fathering a child compatible with an RhD-negative mother.
The desire for noninvasive fetal diagnosis has been encouraged by the detection of fetal cells in maternal circulation (33-35) as well as the detection of fetal DNA in maternal plasma (36,37). However, the low numbers of fetal cells in maternal circulation (38) and their persistence for up to decades after delivery (39) have been an obstacle to their molecular diagnostic utilization. Fetal DNA, on the other hand, has been reported at mean fraction concentrations of 3.4% and 6.2% in maternal plasma during early and late pregnancies, respectively (40), and, furthermore, fetal DNA has also been reported to rapidly clear from maternal plasma, possessing a half-life of only minutes (41). These characteristics make fetal DNA present in maternal plasma an attractive target for prenatal genotyping, and several groups have used this approach for RHD genotyping of fetuses carried by alloimunized RhD-negative mothers (42-45). Collectively, their results indicate that reliable typing can be obtained, especially during and after the second trimester. Realizing the full diagnostic potential of this source of fetal DNA will require sensitive and highly discriminating detection chemistries, because many antigen systems involved in immune cytopenic disorders differ by a single nucleotide, unlike RhD; however, the future appears promising.
2.2. GENOTYPING RHCC AND RHEE RED CELL ANTIGEN SYSTEMS The RHCE gene encodes the peptide carrying the Rh E/e and C/c antigens (46). The RhC/c epitope arises from a single-base substitution within exon 2 at nt307; this substitution results in the incorporation of serine (RhC = CCT) or proline (Rhc = TCT) at amino acid 103 (19,47). There are five additional base substitutions between the RHC and RHc alleles within exons 1 and 2; however, these polymorphisms have been shown not to be involved in the RhC/c serology (48,49). The expression of the C/c antigens is thought to be conformation dependent because the RhD peptide also specifies serine at residue 103 but does not express RhC antigenicity. The RhE/e epitope arises from a single-nucleotide substitution within exon 5 at position 676; this substitution results in the
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