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One-step noninvasive prenatal testing (NIPT) for autosomal recessive homozygous point mutations using digital PCR.

ABSTRACT: Previously, we introduced a noninvasive prenatal testing (NIPT) protocol for diagnosing compound heterozygous autosomal recessive point mutations via maternal plasma DNA and simulated control genomic DNA sampling based on fetal DNA fraction. In our present study, we have improved our NIPT protocol to make it possible to diagnose homozygous autosomal recessive point mutations without the need to acquire fetal DNA fraction. Moreover, chi-squared test and empirical statistical range based on the proportion of mutant allele reads among the total reads served as the gatekeeping method. If this method yielded inconclusive results, then the Bayesian method was performed; final conclusion was drawn from the results of both methods. This protocol was applied to three families co-segregating congenital sensorineural hearing loss with monogenic homozygous mutations in prevalent deafness genes. This protocol successfully predicted the fetal genotypes from all families without the information about fetal DNA fraction using one-step dPCR reactions at least for these three families. Furthermore, we suspect that confirmatory diagnosis under this protocol is possible, not only by using picodroplet dPCR, but also by using the more readily available chip-based dPCR, making our NIPT protocol more useful in the diagnosis of autosomal recessive point mutations in the future.

SUBMITTER: Chang MY

PROVIDER: S-EPMC5811538 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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One-step noninvasive prenatal testing (NIPT) for autosomal recessive homozygous point mutations using digital PCR.

Chang Mun Young MY Ahn Soyeon S Kim Min Young MY Han Jin Hee JH Park Hye-Rim HR Seo Han Kyu HK Yoon Jinsun J Lee Seungmin S Oh Doo-Yi DY Kang Changsoo C Choi Byung Yoon BY

Scientific reports 20180213 1

Previously, we introduced a noninvasive prenatal testing (NIPT) protocol for diagnosing compound heterozygous autosomal recessive point mutations via maternal plasma DNA and simulated control genomic DNA sampling based on fetal DNA fraction. In our present study, we have improved our NIPT protocol to make it possible to diagnose homozygous autosomal recessive point mutations without the need to acquire fetal DNA fraction. Moreover, chi-squared test and empirical statistical range based on the pr ...[more]

PMID: 29440752

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Project description:Background:Noninvasive prenatal testing (NIPT) for fetal antigens is a common standard for targeted immune prophylaxis in RhD-mediated hemolytic disease of the fetus and newborn, and is most frequently done by quantitative PCR (qPCR). A similar approach is considered for other blood group and human platelet alloantigens (HPA). Because of a higher sensitivity compared to qPCR for rare molecule detection, we established and validated digital PCR (dPCR) assays for the detection of RHD exons 3, 5 and 7, KEL1, HPA-1a, and HPA-5b from cell-free DNA (cfDNA) in plasma. The dPCR assays for the Y-chromosomal marker amelogenin and autosomal SNPs were implemented as controls for the proof of fetal DNA. Methods:Validation was performed on dilution series of mixed plasma samples from volunteer donors with known genotypes. After preamplification of the target loci, two-color (FAM and VIC) TaqManTM probe chemistry and chip-based dPCR were applied. The assays for RHD included GAPDH as an internal control. For the diallelic markers KEL1/2, HPA-1a/b, HPA-5a/b, and AMEL-X/Y and 3 autosomal SNPs, the probes enabled allelic discrimination in the two fluorescence channels. The dPCR protocol for NIPT was applied to plasma samples from pregnant women. Results:The RHD exon 5 assay allowed the detection of a 0.05% RHD target in an RhD-negative background, whereas the exon 7 assay required at least a 0.25% target. The exon 3 assay showed the highest background and required at least a 2.5% RHD target for reliable detection. The dPCR assays for the diallelic markers revealed similar sensitivity and enabled the detection of at least a 0.5% target allele. The HPA-1a assay was the most sensitive and allowed target detection in plasma mixtures containing only 0.05% HPA-1a. The plasma samples from 13 pregnant women at different gestational ages showed unambiguous positive and negative results for the analyzed targets. Conclusion:Analysis of cfDNA from maternal plasma using dPCR is suitable for the detection of fetal alleles. Because of the high sensitivity of the assays, the NIPT protocol for RhD, KEL1, and HPA can also be applied to earlier stages of pregnancy.

Dataset Information

One-step noninvasive prenatal testing (NIPT) for autosomal recessive homozygous point mutations using digital PCR.

Publications

One-step noninvasive prenatal testing (NIPT) for autosomal recessive homozygous point mutations using digital PCR.

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