Validation of Extensive Next-Generation Sequencing Method for Monogenic Disorder Analysis on Cell-Free Fetal DNA: Noninvasive Prenatal Diagnosis

J Mol Diagn. 2019 Jul;21(4):572-579. doi: 10.1016/j.jmoldx.2019.02.010. Epub 2019 Apr 25.


During pregnancy, a percentage of the cell-free DNA circulating in the maternal blood is represented by the cell-free fetal DNA (cffDNA), constituting an accessible source for noninvasive prenatal genetic screening. The coexistence of the maternal DNA, the dominant fraction of cell-free DNA, together with the cffDNA component and the scarcity of the cffDNA itself make applying traditional methods of genetics and molecular biology impossible. Next-generation sequencing methods are widely used to study fetal aneuploidies. However, in monogenic disorders, there have been relatively few studies that analyzed single mutations. We present a method for the analysis of an extended group of gene variants associated with recessive and dominant autosomal disorders using next-generation sequencing. The proposed test should allow a complete analysis of common genetic disorders and pathogen-associated variants for diagnostic use. The analysis of cffDNA for single gene disorders may replace invasive prenatal diagnosis methods, associated with the risk of spontaneous abortion and psychological stress for patients. The proposed test should assess reproductive risk for both genetic family disorders and de novo occurrences of the disease. The application of this method to a case of beta-thalassemia is also discussed.

MeSH terms

  • Alleles
  • Cell-Free Nucleic Acids*
  • Female
  • Genetic Diseases, Inborn / diagnosis*
  • Genetic Diseases, Inborn / genetics*
  • Genetic Markers*
  • Genetic Predisposition to Disease
  • Genomics / methods
  • High-Throughput Nucleotide Sequencing* / methods
  • Humans
  • Noninvasive Prenatal Testing* / methods
  • Pregnancy
  • Reproducibility of Results
  • Software


  • Cell-Free Nucleic Acids
  • Genetic Markers