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

Area of Science:

• Genetics
• Molecular Biology
• Biochemistry

Background:

• Non-invasive prenatal testing (NIPT) for fetal aneuploidies is widely used.
• Current NGS methods require at least 4% fetal fraction for reliable NIPT.
• Inaccurate fetal fraction assessment increases misdiagnosis risk in NIPT.

Purpose of the Study:

• To develop a novel assay for accurate fetal fraction estimation in maternal plasma.
• To improve the reliability of NIPT by addressing limitations in fetal fraction assessment.

Main Methods:

• Identified fetal-specific differentially methylated regions (DMRs) using MSRD, MeDIP, and NGS.
• Developed a multiplex ddPCR assay incorporating seven selected DMRs.
• Validated the assay against a Chromosome Y multiplex ddPCR assay in male pregnancies.

Main Results:

• A model using four DMRs (FFMM) was developed for fetal fraction estimation.
• High correlation (r=0.86) was observed between FFMM and YMM-based fetal fractions in 85 male pregnancies.
• The FFMM model was further validated in an independent set of 53 male pregnancies.

Conclusions:

• Developed a SNP-, sex-, and ploidy-independent methylation-based multiplex ddPCR assay.
• This assay provides accurate fetal fraction estimation for NIPT.
• The novel method enhances the accuracy and reliability of non-invasive prenatal testing.