Detection of Paternal IVS-II-1 (G>A) (HBB: c.315+1G>A) Mutation in Cell-Free Fetal DNA Using COLD-PCR assay

Standardization of noninvasive prenatal diagnosis (PND) method that can identify common mutations in the population is of great value. The purpose of this study was to find the paternal HBB gene IVS-II-1 (G>A) (HBB: c.315+1G>A) mutation in maternal plasma cell-free DNA using the co-amplification at lower denaturation temperature-polymerase chain reaction (COLD-PCR) method. We designed simulated circulating free DNA (cfDNA) in maternal plasma to optimize the COLD-PCR assay. Peripheral blood samples were collected from normal and IVS-II-1 heterozygous individuals as well as five heterozygous pregnant women whose husbands were carriers of IVS-II-1. The cfDNA was extracted from the plasma and subjected to optimized COLD-PCR followed by Sanger sequencing. The optimized protocol was tested on simulated cfDNA samples with proportions of 8.0, 6.0, 4.0 and 2.0%, and the results showed that the COLD-PCR is informative on samples containing 8.0% mutant alleles and above. The patients were undergoing invasive PND procedures via chorionic villi sampling (CVS) as scheduled at the 12th week of gestation. Paternal IVS-II-1 was detected in cfDNA samples of three patients who were in complete concordance with the outcome of CVS. Despite the limitations of the COLD-PCR method in noninvasive PND, it can be considered as a cost-effective screening option. The use of this approach for screening at-risk patients can prevent unnecessary invasive procedures identifying common mutations in high-prevalence diseases.