GENOTYPING OF RHD BY MULTIPLEX POLYMERASE-CHAIN-REACTION ANALYSIS OF 6 RHD-SPECIFIC EXONS

BACKGROUND: Qualitative RHD variants are the result of the replacement of RHD exons by their RHCE counterparts or of point mutations in RHD causing amino acid substitutions. For RHD typing, the use of at least two RHD typing polymerase chain reaction (PGR) assays directed at diff erent regions of RHD is advised to prevent discrepancies between pheno typing and genotyping results, but even then discrepancies occur. A mu ltiplex RHD PCR based on amplification of six RHD-specific exons in on e reaction mixture is described. STUDY DESIGN AND METHODS: Six RHD-spe cific primer sets were designed to amplify RHD exons 3, 4, 5, 6, 7, an d 9. DNA from 119 donors (87 D+, 14 D- and 18 with known D variants; w hites and nonwhites) with known Rh phenotypes was analyzed. RESULTS: A ll six RHD-specific exons from 85 D+ individuals were amplified, where as none of the RHD exons from 13 D- individuals were amplified. Multip lex PGR analysis showed that the genotypes of two donors typed as D+ w ere D-IVa and D-Va. Red cell typing confirmed these findings. From all D variants tested (D-IIIa D-IVa, D-IVb, D-Va, D-VI D-DFR, D-DBT) and from R-o(Har): RHD-specific exons were amplified as expected from the proposed genotypes. CONCLUSION: The multiplex PCR assay is reliable in determining genotypes in people who have the D+ and partial D phenoty pes as well as in discovering people with new D variants. Because the multiplex PCR is directed at six regions of RHD, the chance of discrep ancies is markedly reduced. The entire analysis can be performed in on e reaction mixture, which results in higher speed, higher accuracy, an d the need for smaller samples. This technique might be of great value in prenatal RHD genotyping.