Segmental duplication as potential biomarkers for non-invasive prenatal testing of aneuploidies

Abstract

Background: Segmental duplication (SD) regions are distinct targets for aneuploidy detection owing to the virtual elimination of amplification bias. The difficulty of searching SD sequences for assay design has hampered their applications. Methods: We developed a computational program, ChAPDes, which integrates SD searching, refinement, and design of specific PCR primer/probe sets in a pipeline to remove most of the manual work. The generated primer/probe sets were first tested in a multiplex multicolour melting curve analysis for the detection of five common aneuploidies. The primer/probe sets were then tested in a digital PCR assay for the detection of trisomy 21. Finally, a digital PCR protocol was established to quantify maternal plasma DNA sequences for the non-invasive prenatal detection of fetal trisomy 21. Findings: ChAPDes could output 21,772 candidate primer/probe sets for trisomy 13, 18, 21 and sex chromosome aneuploidies within 2 working days. Clinical evaluation of the multiplex multicolour melting curve analysis involving 463 fetal genomic DNA samples revealed a sensitivity of 100% and specificity of 99.64% in comparison with the reference methods. Using the established digital PCR protocol, we correctly identified two trisomy 21 fetuses and thirteen euploid foetuses from the maternal plasma samples. Interpretation: The combination of ChAPDes with digital PCR detection could facilitate the use of SD as potential biomarkers for the non-invasive prenatal testing of fetal chromosomal aneuploidies.

Multiplex Asymmetric PCR by Combining the Amplification Refractory Mutation System with the Homo-Tag-Assisted Nondimer System

“…This was demonstrated through the genotyping of 11 biallelic SNPs using MMCA, where a sample containing 20% trisomy 21 could be discriminated from a normal sample. Such distinct allele quantification was reproducible when tested with gDNA from different sources and extraction methods, factors that might otherwise affect the reproducibility of other quantitative approaches for copy number detection . The quantitative feature of A-H PCR was successfully used to design a trisomy 21 assay, with the correct identification of 31 trisomy 21 cases in 342 clinical samples, A-H PCR improved the number of quantitative SNP loci in a single reaction from two to 11 in our study.…”

A dPCR-NIPT assay for detections of trisomies 21, 18 and 13 in a single-tube reaction-could it replace serum biochemical tests as a primary maternal plasma screening tool?

“…After PCR, the proportion of positive partitions is applied to accurately quantify the target concentration using Poisson’s statistics [ 36 – 38 ]. There are quite a few publications demonstrated the usefulness of dPCR technology in detection of fetal T21 from variety of angles, some used the earlier versions of dPCR platforms or invasive CVS/AS samples to prove the concept [ 25 , 39 , 40 ], while others evaluated the feasibilities in detections of fetal T21 and T18 in maternal plasma samples [ 41 – 44 ].…”