Development of a new methylation-based fetal fraction estimation assay using multiplex ddPCR

Background: Non-invasive prenatal testing (NIPT) for fetal aneuploidies has rapidly been incorporated into clinical practice. Current NGS-based methods can reliably detect fetal aneuploidies non-invasively with fetal fraction of at least 4%. Inaccurate fetal fraction assessment can compromise the accuracy of the test as affected samples with low fetal fraction have an increased risk for misdiagnosis. Using a novel set of fetal-specific differentially methylated regions (DMRs) and methylation sensitive restriction digestion (MSRD), we developed a multiplex ddPCR assay for accurate detection of fetal fraction in maternal plasma. Methods: We initially performed MSRD followed by methylation DNA immunoprecipitation (MeDIP) and NGS on fetal and non-pregnant female tissues to identify fetal-specific DMRs. DMRs with the highest methylation difference between the two tissues were selected for fetal fraction estimation employing MSRD and multiplex ddPCR. Chromosome Y multiplex ddPCR assay (YMM) was used as a reference standard, to develop our fetal fraction estimation model in male pregnancy samples. Additional 123 samples were tested to examine whether the model is sex dependent and/or ploidy dependent. Results: In all, 93 DMRs were identified of which seven were selected for fetal fraction estimation. Statistical analysis resulted in the final model which included four DMRs (FFMM). High correlation with YMM-based fetal fractions was observed using 85 male pregnancies (r = 0.86 95% CI: 0.80–0.91). The model was confirmed using an independent set of 53 male pregnancies. Conclusion: By employing a set of well-characterized DMRs, we developed a SNP-, sex- and ploidy-independent methylation-based multiplex ddPCR assay for accurate fetal fraction estimation.