C A C A T

Exon 1

Kxon 2

Exon 5

Exon 1

Kxon 2

Exon 5

Fig. 2. (A) Nucleotide differences between the RHCE and RHD genes. Numbers indicate nucleotide positions, and epitope-related positions are illustrated in black. Note that, for simplicity, only RHCE and RHce haplotypes are shown (RhcE and RHCe haplotypes exist). (B) Strategy for typing RHCc/Ee alleles by ASPCR targeting nucleotides 48, 307, and 676. (C) RHC/c genotyping by ASPCR and 2% agarose gel electrophoresis. Products of each analysis are run in sets of three; lane 1: the RHC intron 2 reaction; lane 2: the RHC nt48 cytosine reaction; lane 3: the RHc nt308 cytosince reaction. The RhC intron 2 reaction produces a 594-bp product if the RhC allele is present and a 485-bp product from either RHc or RHD. In the RhC nt48 cytosine reaction and the Rhc nt308 cytosine reaction, a 423-bp HGH control product is detected in each reaction possessing the DNA template, whereas the RHC nt48 amplicon generates a 114-bp product and the RHc nt 308 amplicon generates a 179-bp product. Panel A: normal RhC homozygotes; panel B: normal Rhc homozygotes; panel C: serological RhCc heterozygote which lacks the intron 2 insertion (RhC false-negative); panel D: serological Rhcc homozygous sample which possesses cytosine at nt48 (RhC false-positive). Lane S: 100-bp DNA ladder (Gibco/BRL, Gaithersburg MD).

Fig. 2. (A) Nucleotide differences between the RHCE and RHD genes. Numbers indicate nucleotide positions, and epitope-related positions are illustrated in black. Note that, for simplicity, only RHCE and RHce haplotypes are shown (RhcE and RHCe haplotypes exist). (B) Strategy for typing RHCc/Ee alleles by ASPCR targeting nucleotides 48, 307, and 676. (C) RHC/c genotyping by ASPCR and 2% agarose gel electrophoresis. Products of each analysis are run in sets of three; lane 1: the RHC intron 2 reaction; lane 2: the RHC nt48 cytosine reaction; lane 3: the RHc nt308 cytosince reaction. The RhC intron 2 reaction produces a 594-bp product if the RhC allele is present and a 485-bp product from either RHc or RHD. In the RhC nt48 cytosine reaction and the Rhc nt308 cytosine reaction, a 423-bp HGH control product is detected in each reaction possessing the DNA template, whereas the RHC nt48 amplicon generates a 114-bp product and the RHc nt 308 amplicon generates a 179-bp product. Panel A: normal RhC homozygotes; panel B: normal Rhc homozygotes; panel C: serological RhCc heterozygote which lacks the intron 2 insertion (RhC false-negative); panel D: serological Rhcc homozygous sample which possesses cytosine at nt48 (RhC false-positive). Lane S: 100-bp DNA ladder (Gibco/BRL, Gaithersburg MD).

incorporation of proline (RHE = CCT) for alanine (Rhe = GCT) at amino acid 226 (19,47).

The homology between the RHCE and RHD genes again presents an obstacle when attempting to genotype the RhCc system. However, it is possible to design allele-specific primers for RHc, RHE, and RHe that target the epitope-specifying nucleotide (Fig. 2). When genotyping RHc utilizing nt307 in our laboratory, complete correlation was observed between the presence of cyto-sine at nt307 and Rhc serology in 282 individuals of Caucasian and African-American ancestry (14). Unfortunately, it is not possible to selectively amplify RHC in the presence of RHD using the epitope-specific polymorphism at position 307 because the RHC

allele is identical to RhD within exon 2 (50,51). Hyland et al. (48) and Wolter et al. (49) reported that the C to G transition at nt 48 within exon 1 correlated well with RhC or Rhc serological typing, respectively; however, approx 5% of the RHc alleles mimicked RHC in that they possessed cytosine at nucleotide 48. In a study with African blacks, 74% of Rhcc individuals contained a cytosine at nucleotide 48 (49). We have observed a 7.2% (4/55) false-heterozygous rate when genotyping serologically typed Rhcc Caucasian individuals (14), which is consistent with previous reports (48,49,52,53). However, we have also observed a 56.3% (45/80) false-heterozygous rate when genotyping serologically typed Rhcc African-American individuals, which drastically decreases the utility of this polymorphic position for RHC geno-typing within this ethnic group. No false-negative genotyping results were observed when using the RhC-associated nt48 cyto-sine in either African-Americans or Caucasians. Therefore, this locus can be cautiously used to establish the fetal absence of the RHC allele in Rhcc mothers sensitized to RhC, with the recognition that there is the possibility of a false-heterozygous genotyping of the fetus.

A unique 109-bp insertion within intron 2 of the RHCE gene has been correlated with RhC serology in two independent studies that collectively evaluated over 600 individuals with complete concordance (52,54). In our laboratory, primers flanking the RHC-specific intron 2 insertion were used to genotype 282 serotyped individuals (128 African-Americans, 154 Caucasians). Among 154 Caucasian samples, an overall 100% concordance rate was observed between serology and presence or absence of the RhC-associated intron 2 insertion, and complete resolution of the 4 serological Rhcc Caucasian nucleotide 48 false-positive samples was observed (14). Among African-Americans, no false-positives were observed when utilizing the RhC-associated intron 2 insertion, and complete resolution of the 45 serological Rhcc African-American nucleotide 48 false-positive samples was observed. However, a 23.9% (11/46) false-negative rate was detected when genotyping serotyped heterozygotes. It is for this reason this polymorphism cannot be used independently for indirect genotyping of the RHC allele; however, this polymorphism can, in certain circumstances, be useful for use in tandem with the exon 1 nt48 cytosine for RHC genotyping. Tax et al. has recently reported a new strategy for more accurate genotyping RHC alleles through selective amplification of RHC variants and has observed >99% concordance with serology across 1071 individuals of different racial/ethnic groups (55). The RhEe system arises through a single-point mutation within exon 5 (Fig. 2) and genotyping can be accomplished without complications arising from RhD (50,51).

The Rh system is complex and highlights some of the caveats associated with using DNA-based reference tests. In order to identify cases where allelic variants exist, regardless of the antigen system in question, it is advisable to always establish concordance between the serotypes and genotypes of the parents before predicting fetal phenotype based on a fetal genotyping result.

Pregnancy Guide

Pregnancy Guide

A Beginner's Guide to Healthy Pregnancy. If you suspect, or know, that you are pregnant, we ho pe you have already visited your doctor. Presuming that you have confirmed your suspicions and that this is your first child, or that you wish to take better care of yourself d uring pregnancy than you did during your other pregnancies; you have come to the right place.

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