Digital PCR for the molecular detection of fetal chromosomal aneuploidy

Abstract

Trisomy 21 is the most common reason that women opt for prenatal diagnosis. Conventional prenatal diagnostic methods involve the sampling of fetal materials by invasive procedures such as amniocentesis. Screening by ultrasonography and biochemical markers have been used to risk-stratify pregnant women before definitive invasive diagnostic procedures. However, these screening methods generally target epiphenomena, such as nuchal translucency, associated with trisomy 21. It would be ideal if noninvasive genetic methods were available for the direct detection of the core pathology of trisomy 21. Here we outline an approach using digital PCR for the noninvasive detection of fetal trisomy 21 by analysis of fetal nucleic acids in maternal plasma. First, we demonstrate the use of digital PCR to determine the allelic imbalance of a SNP on PLAC4 mRNA, a placenta-expressed transcript on chromosome 21, in the maternal plasma of women bearing trisomy 21 fetuses. We named this the digital RNA SNP strategy. Second, we developed a nonpolymorphism-based method for the noninvasive prenatal detection of trisomy 21. We named this the digital relative chromosome dosage (RCD) method. Digital RCD involves the direct assessment of whether the total copy number of chromosome 21 in a sample containing fetal DNA is overrepresented with respect to a reference chromosome. Even without elaborate instrumentation, digital RCD allows the detection of trisomy 21 in samples containing 25% fetal DNA. We applied the sequential probability ratio test to interpret the digital PCR data. Computer simulation and empirical validation confirmed the high accuracy of the disease classification algorithm.

Fig. 1.

Illustration of the analytical steps in digital RNA SNP and digital RCD analyses for T21 detection. Only a representative 96-well subset of the 384-well data is shown for one euploid and one T21 case for each of digital RNA SNP and digital RCD analyses, respectively. The T21 data depicted in the digital RNA SNP experiment represent a case where the G allele is overrepresented, i.e., a fetal genotype of AGG.

Fig. 2.

SPRT analysis. (A) A pair of SPRT curves delimits the decision boundaries for accepting or rejecting the hypotheses that the sample belonged to a euploid or aneuploid fetus. (B) The decision boundaries of the SPRT curves would vary according to the template concentration. Curves applicable to digital RNA SNP analysis are shown.

Fig. 3.

SPRT interpretation of digital RCD analyses. (A) Placental DNA samples. (B) DNA mixtures of 50% placenta/maternal buffy coat. (C) DNA mixtures of 25% placenta/maternal buffy coat. Numbers at the top of B and C indicate the number of 384-well plates required before the data set was classifiable for the cases delimited by the dotted lines surrounding each number.