Rapid Zygosity Determination Using Digital PCR.

BACKGROUND

Paternal zygosity testing is used for determining homo- or hemizygosity of in pregnancies that are at a risk of hemolytic disease of the fetus and newborn. At present, this is achieved by using real-time PCR or the PCR, which can be difficult to interpret and unreliable, particularly for black African populations.

METHODS

DNA samples extracted from 53 blood donors were analyzed using 2 multiplex reactions for -specific targets against a reference () to determine gene dosage by digital PCR. Results were compared with serological data, and the correct genotype for 2 discordant results was determined by long-range PCR (LR-PCR), next-generation sequencing, and conventional Sanger sequencing.

RESULTS

The results showed clear and reliable determination of zygosity using digital PCR and revealed that 4 samples did not match the serologically predicted genotype. Sanger sequencing and long-range PCR followed by next-generation sequencing revealed that the correct genotypes for samples 729M and 351D, which were serologically typed as RR (DCe/DcE), were Rr' (DcE/dCe) for 729M and Rr″ (DCe/dcE), Rr (Dce/dCE), or Rr (DCE/dce) for 351D, in concordance with the digital PCR data.

CONCLUSIONS

Digital PCR provides a highly accurate method to rapidly define blood group zygosity and has clinical application in the analysis of Rh phenotyped or genotyped samples. The vast majority of current blood group genotyping platforms are not designed to define zygosity, and thus, this technique may be used to define paternal zygosity in pregnancies that are at a risk of hemolytic disease of the fetus and newborn and can distinguish between homo- and hemizygous -positive individuals.