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

Abstract

ObjectiveTo 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.MethodsWe 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.ResultsUsing 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.ConclusionsOur 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.

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

“…Selective denaturation stage (Tc) is vital for fast COLD-PCR to precisely denature the mutated sequence ( 14 , 23 ). First, 10 rounds of conventional PCR were performed to amplify and generate a sufficient template for COLD-PCR.…”

“…There are several forms of COLD-PCR, among them, fast COLD-PCR is cheapest and easiest. Fast COLD-PCR is a modified form of conventional PCR involving an additional parameter, the critical denaturation temperature (Tc), primarily suitable for Tm-reducing mutations (for example, G:C>A:T or G:C>T:A) ( 14 ). Following this selective denaturation, only mutant (MT) A/T-containing alleles are obtained and wild-type (WT) G/C alleles are left double-stranded (ds).…”

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

“…Co-amplification at lower denaturation temperature-PCR (COLD-PCR) followed by Sanger sequencing or NGS is a method that takes advantage of PCR amplification bias to preferentially amplify and detect minority alleles based on differences in denaturation temperature of the reference and alternate DNA sequences [Figure 1C]. Galbiati et al assessed the use of COLD-PCR followed by Sanger sequencing for NIPD of several common variants (down to a level of < 1%) causative of β-thalassaemia [46] and cystic fibrosis [47] , as well as a family-specific 18 bp deletion in TWIST1, causative of craniosynostosis [48] . This approach was validated against highly sensitive microarray assays [47] and has also been applied in the research setting for NIPD of paternally inherited mutations causative of β-thalassaemia (HBB) [49,50] , and to detect feto-maternal platelet incompatibility [51] .…”

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

“…NIPT techniques independent of NGS are restricted to the identification of alleles which are present in the foetal genome but not in the maternal one-for example RHD (Rh blood group, D antigen) genotyping in RhD-negative mothers 13 , detection of mutant paternal alleles in families with monogenic disorders, such as achondroplasia 14 or early onset primary dystonia 15 , and the exclusion of affected status in autosomal recessive disorders, such as cystic fibrosis 16 or β-thalassemia 17 in families where the parents have different mutations and therefore the paternal allele can be determined on the maternal background.…”