Noninvasive prenatal diagnosis of beta-thalassemia disease by using digital PCR analysis of cell-free fetal DNA in maternal plasma

Introduction: Prenatal diagnosis of thalassemia disease usually based on invasive technique. Non-invasive diagnosis using cell-free fetal DNA (cff-DNA) were described with various laboratory technique. The aim of this study to identify the performance of dPCR for analyzing cff-DNA in maternal plasma to diagnose fetal beta-thalassemia diseases.
Methods: Thirty-five couples at risk of fetal beta-thalassemia disease caused by four common mutations of HBB were enrolled at 12-18 weeks. The dPCR assay was designed to detect and quantify paternally-inherited beta-thalassemia allele (PIB-M) and maternally-inherited beta-thalassemia allele (MIB-M) from cff-DNA in maternal plasma. 
Results: Of 29 couples with different paternal/maternal mutations, all cases who inherited paternal mutation had detectable PIB-M. The MIB-mutant/wild-type (MIB-M/MIB-N) ratio in the mothers whose fetuses did not inherit maternal mutation was 0.87+/-0.07 which was significantly lower than that of the mothers whose fetuses inherited maternal mutation, 1.01+/-0.05. The sensitivity and specificity of MIB-M/MIB-N ratio >0.95 in predicting fetus inheriting maternal mutation was 100 and 92.3%, respectively. In four couples with same paternal/maternal mutation, IB-M/IB-N ratio of >0.95 correctly predicted the presence of an inheritance of at least one beta-thalassemia allele. In two couples with paternal Hb E/beta-thalassemia, the presence of PIB-M and the MIB-M/MIB-N ratio of >0.95 correctly predicted the presence of paternal/maternal mutations, respectively.
Conclusions: The method of analyzing cff-DNA in maternal plasma by dPCR is efficient for prenatal diagnosis of beta-thalassemia.
