Establishment of a medium‐throughput approach for the genotyping of RHD variants and report of nine novel rare alleles

Background The routinely used serologic methods are robust in accurately typing standard D − or D + blood. However, they result in discrepancy in weak or partial D blood, which requires genetic analysis. We have previously used denaturing high‐performance liquid chromatography ( DHPLC ) to screen the entire RHD ‐coding sequence. However, DHPLC is technically challenging, labor‐intensive, and time‐consuming. To overcome these inconveniences, we sought to develop a new two‐step approach. Study Design and Methods A total of 430 blood samples with D phenotype ambiguity were recruited for this study. The three most frequent weak D alleles (i.e., weak D , Type 1; weak D , Type 2; and weak D , Type 3), which altogether account for 60% to 90% of the atypical RHD alleles in the C aucasian population, were first identified by T m‐shift genotyping. The remaining unidentified samples were then subjected to a single‐tube multiplex polymerase chain reaction ( PCR ) amplification of all 10 RHD exons followed by direct sequencing. Results Optimal conditions for efficient and reliable identification of the three most common weak D variants by T m‐shift genotyping were established. All 10 RHD exons were successfully amplified in a single‐multiplex PCR procedure. Employment of the two‐step analysis identified RHD variants in 91.6% of the 430 studied samples. Two of the nine previously undescribed variants, c .335 G > T and c .939 G > A , were found to cause aberrant mRNA splicing by means of a splicing minigene assay. Conclusion The new two‐step analysis proved to be much easier and cheaper than the DHPLC method and therefore is convenient to be used as a routine, medium‐throughput approach for RHD genotyping.