Fast Temperature-Gradient COLD PCR for the Enrichment of the Paternally Inherited SNPs in Cell Free Fetal DNA; an Application to Non-invasive Prenatal Diagnosis of β-thalassaemia

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2018 Jul 26

PMID 30044883

Citations 4

Authors

Stefania Byrou

G Mike Makrigiorgos

Agathoklis Christofides

Ioannis Kallikas

Thessalia Papasavva

Marina Kleanthous

Affiliations

Soon will be listed here.

Abstract

Objective: To develop a sensitive, specific, simple, cost-effective and reproducible platform for the non-invasive prenatal detection of paternally inherited alleles for β-thalassaemia. The development of such an assay is of major significance in order to replace currently-applied invasive methods containing inherent fetal loss risks.

Methods: We present a fast Temperature-Gradient Co-amplification at Lower Denaturation Temperature Polymerase Chain Reaction (fast TG COLD PCR) methodology for the detection of the paternally-inherited fetal alleles in maternal plasma. Two single-nucleotide polymorphisms (SNPs), rs7480526 (G/T) and rs968857 (G/A) that are located on the β-globin gene cluster and exhibit a high degree of heterozygosity in the Cypriot population were selected for evaluation. Seventeen maternal plasma samples from pregnancies at risk for β-thalassemia were analysed for the selected SNPs using the novel fast TG COLD PCR assay.

Results: Using fast TG COLD PCR, the paternally inherited allele in cell free fetal DNA was correctly determined for all the 17 maternal plasma samples tested, showing full agreement with the Chorionic Villus Sampling (CVS) analysis.

Conclusions: Our findings are encouraging and demonstrate the efficiency and sensitivity of fast TG COLD PCR in detecting the minor paternally-inherited fetal alleles in maternal plasma for the development of a NIPD assay for β-thalassaemia.

Citing Articles

Non-Invasive Determination of the Paternal Inheritance in Pregnancies at Risk for β-Thalassaemia by Analyzing Cell-Free Fetal DNA Using Targeted Next-Generation Sequencing.

Byrou S, Brouwer R, Tomazou M, Tamana S, Kountouris P, Lederer C Int J Mol Sci. 2025; 26(2).

PMID: 39859286 PMC: 11765003. DOI: 10.3390/ijms26020570.

Non-invasive prenatal diagnosis (NIPD): current and emerging technologies.

Hanson B, Paternoster B, Povarnitsyn N, Scotchman E, Chitty L, Chandler N Extracell Vesicles Circ Nucl Acids. 2024; 4(1):3-26.

PMID: 39698301 PMC: 11648410. DOI: 10.20517/evcna.2022.44.

Introduction of new alternative pipeline using multiplexed fast COLD‑PCR together with sequencing approach highlighting pharmacoeconomics by detection of variants.

Nandar Y, Duangmano S, Lucksiri A, Sirikul C, Palacajornsuk P, Anukul N Biomed Rep. 2023; 17(6):99.

PMID: 36606140 PMC: 9808490. DOI: 10.3892/br.2022.1582.

The current applications of cell-free fetal DNA in prenatal diagnosis of single-gene diseases: A review.

Mortazavipour M, Mahdian R, Shahbazi S Int J Reprod Biomed. 2022; 20(8):613-626.

PMID: 36313257 PMC: 9596931. DOI: 10.18502/ijrm.v20i8.11751.

Detection of cell-free foetal DNA fraction in female-foetus bearing pregnancies using X-chromosomal insertion/deletion polymorphisms examined by digital droplet PCR.

Zednikova I, Pazourkova E, Lassakova S, Vesela B, Korabecna M Sci Rep. 2020; 10(1):20036.

PMID: 33208834 PMC: 7676229. DOI: 10.1038/s41598-020-77084-0.

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