Noninvasive Prenatal Diagnosis of Single-Gene Disorders by Use of Droplet Digital PCR

Clin Chem. 2018 Feb;64(2):336-345. doi: 10.1373/clinchem.2017.278101. Epub 2017 Nov 2.


Background: Prenatal diagnosis in pregnancies at risk of single-gene disorders is currently performed using invasive methods such as chorionic villus sampling and amniocentesis. This is in contrast with screening for common aneuploidies, for which noninvasive methods with a single maternal blood sample have become standard clinical practice.

Methods: We developed a protocol for noninvasive prenatal diagnosis of inherited single-gene disorders using droplet digital PCR from circulating cell-free DNA (cfDNA) in maternal plasma. First, the amount of cfDNA and fetal fraction is determined using a panel of TaqMan assays targeting high-variability single-nucleotide polymorphisms. Second, the ratio of healthy and diseased alleles in maternal plasma is quantified using TaqMan assays targeting the mutations carried by the parents. Two validation approaches of the mutation assay are presented.

Results: We collected blood samples from 9 pregnancies at risk for different single-gene disorders, including common conditions and rare metabolic disorders. We measured cases at risk of hemophilia, ornithine transcarbamylase deficiency, cystic fibrosis, β-thalassemia, mevalonate kinase deficiency, acetylcholine receptor deficiency, and DFNB1 nonsyndromic hearing loss. We correctly differentiated affected and unaffected pregnancies (2 affected, 7 unaffected), confirmed by neonatal testing. We successfully measured an affected pregnancy as early as week 11 and with a fetal fraction as low as 3.7% (0.3).

Conclusions: Our method detects single-nucleotide mutations of autosomal recessive diseases as early as the first trimester of pregnancy. This is of importance for metabolic disorders in which early diagnosis can affect management of the disease and reduce complications and anxiety related to invasive testing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Alleles
  • Cell-Free Nucleic Acids / blood
  • Clinical Protocols
  • Female
  • Fetus / metabolism
  • Genes, Recessive
  • Genetic Diseases, Inborn / blood
  • Genetic Diseases, Inborn / diagnosis*
  • Genetic Diseases, Inborn / genetics
  • Genetic Diseases, X-Linked / blood
  • Genetic Diseases, X-Linked / diagnosis
  • Genetic Diseases, X-Linked / genetics
  • Heterozygote
  • Humans
  • Mutation
  • Polymerase Chain Reaction / methods*
  • Polymorphism, Single Nucleotide
  • Pregnancy
  • Prenatal Diagnosis / methods*
  • Reproducibility of Results


  • Cell-Free Nucleic Acids