Development of a new methylation‐based fetal fraction estimation assay using multiplex ddPCR. Issue 2 (10th December 2019)

• Record Type: Journal Article
• Authors: Ioannides, Marios; Achilleos, Achilleas; Kyriakou, Skevi; Kypri, Elena; Loizides, Charalambos; Tsangaras, Kyriakos; Constantinou, Louiza; Koumbaris, George; Patsalis, Philippos C.
• Is Part Of: Molecular genetics & genomic medicine. Volume 8:Issue 2(2020)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2019-12-10
• Subjects: differentially methylated regions -- fetal fraction estimation -- multiplex ddPCR -- non‐invasive prenatal testing; Medical genetics -- Periodicals; Genomics -- Periodicals; 616.042
• DOI: 10.1002/mgg3.1094
• Languages: English
• ISSNs: 2324-9269

Abstract

Abstract: 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. Abstract : We have developed a multiplex droplet digital PCR (ddPCR) assay for accurate fetal fraction estimation using methylation sensitive restriction enzymes (MSREs) and a robust set of novel fetal‐specific differentially methylated regions (DMRs). Clinical implementation of our assay as a first‐tier fetal fraction estimation in NIPT can minimize the possibility of incorrect calls due to low fetal fraction estimation.