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

Transfusion. 2013 Aug;53(8):1821-8. doi: 10.1111/trf.12009. Epub 2012 Dec 11.


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 Caucasian population, were first identified by Tm-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 Tm-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.335G>T and c.939G>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.

Publication types

  • Clinical Trial

MeSH terms

  • Alleles*
  • Base Sequence
  • Cohort Studies
  • Genes, Reporter
  • Genetic Markers
  • Genotyping Techniques*
  • Humans
  • Molecular Sequence Data
  • Multiplex Polymerase Chain Reaction
  • Polymorphism, Single Nucleotide
  • Prospective Studies
  • Reverse Transcriptase Polymerase Chain Reaction
  • Rh-Hr Blood-Group System / genetics*
  • Sequence Analysis, DNA


  • Genetic Markers
  • Rh-Hr Blood-Group System

Associated data

  • GENBANK/JQ405074
  • GENBANK/JQ405075
  • GENBANK/JQ405076
  • GENBANK/JQ405078
  • GENBANK/JQ405079
  • GENBANK/JQ405080
  • GENBANK/JQ405082
  • GENBANK/JQ405083
  • GENBANK/JQ608469