Development of novel noninvasive prenatal testing protocol for whole autosomal recessive disease using picodroplet digital PCR

We developed a protocol of noninvasive prenatal testing (NIPT), employing a higher-resolution picodroplet digital PCR, to detect genetic imbalance in maternal plasma DNA (mpDNA) caused by cell-free fetal DNA (cffDNA). In the present study, this approach was applied to four families with autosomal recessive (AR) congenital sensorineural hearing loss. First, a fraction of the fetal DNA in mpDNA was calculated. Then, we made artificial DNA mixtures (positive and negative controls) to simulate mpDNA containing the fraction of cffDNA with or without mutations. Next, a fraction of mutant cluster signals over the total signals was measured from mpDNA, positive controls, and negative controls. We determined whether fetal DNA carried any paternal or maternal mutations by calculating and comparing the sum of the log-likelihood of the study samples. Of the four families, we made a successful prediction of the complete fetal genotype in two cases where a distinct cluster was identified for each genotype and the fraction of cffDNA in mpDNA was at least 6.4%. Genotyping of only paternal mutation was possible in one of the other two families. This is the first NIPT protocol potentially applicable to any AR monogenic disease with various genotypes, including point mutations.