Background: Non-invasive prenatal diagnosis has found application in a limited number of genetic diseases due to the difficulty in detecting a few copies of fetal mutated sequences in the presence of a large excess of wild-type maternal alleles, even in the case of single-base mutations. Methods: We developed conditions for the enrichment of fetal mutated alleles in maternal plasma based on COamplification at lower denaturation temperature-PCR (COLD-PCR). In particular, we applied a full COLD-PCR protocol to the identification of a p.A87-G92del mutation in the TWIST1 gene causing craniosynostosis in a couple at risk for the disease. Results: The use of the COLD-PCR protocol coupled with direct sequencing enabled correct identification of the fetal paternally inherited mutated allele, in accordance with the result obtained on DNA extracted from chorionic villi. Conclusions: COLD-PCR proved to be a simple and powerful tool for the identification of minority mutated alleles even in the case of a moderately large deletion (18 bp) and confirmed to be very suitable for non-invasive prenatal diagnosis of a variety of genetic diseases.
- free fetal DNA in maternal plasma
- full COLD-PCR
- genetic diseases
- non-invasive prenatal diagnosis
ASJC Scopus subject areas
- Clinical Biochemistry
- Biochemistry, medical