Noninvasive Fetal Genotyping by Droplet Digital PCR to Identify Maternally Inherited Monogenic Diabetes Variants

Clin Chem. 2020 Jul 1;66(7):958-965. doi: 10.1093/clinchem/hvaa104.


Background: Babies of women with heterozygous pathogenic glucokinase (GCK) variants causing mild fasting hyperglycemia are at risk of macrosomia if they do not inherit the variant. Conversely, babies who inherit a pathogenic hepatocyte nuclear factor 4α (HNF4A) diabetes variant are at increased risk of high birth weight. Noninvasive fetal genotyping for maternal pathogenic variants would inform pregnancy management.

Methods: Droplet digital PCR was used to quantify reference and variant alleles in cell-free DNA extracted from blood from 38 pregnant women heterozygous for a GCK or HNF4A variant and to determine fetal fraction by measurement of informative maternal and paternal variants. Droplet numbers positive for the reference/alternate allele together with the fetal fraction were used in a Bayesian analysis to derive probability for the fetal genotype. The babies' genotypes were ascertained postnatally by Sanger sequencing.

Results: Droplet digital PCR assays for GCK or HNF4A variants were validated for testing in all 38 pregnancies. Fetal fraction of ≥2% was demonstrated in at least 1 cell-free DNA sample from 33 pregnancies. A threshold of ≥0.95 for calling homozygous reference genotypes and ≤0.05 for heterozygous fetal genotypes allowed correct genotype calls for all 33 pregnancies with no false-positive results. In 30 of 33 pregnancies, a result was obtained from a single blood sample.

Conclusions: This assay can be used to identify pregnancies at risk of macrosomia due to maternal monogenic diabetes variants.

Keywords: Bayesian analysis; droplet digital PCR; monogenic diabetes; non-invasive prenatal diagnosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers / blood
  • DNA / blood*
  • Diabetes Mellitus / enzymology
  • Diabetes Mellitus / genetics*
  • Female
  • Fetal Macrosomia / diagnosis
  • Fetal Macrosomia / genetics
  • Fetus
  • Genotype
  • Genotyping Techniques / methods
  • Genotyping Techniques / statistics & numerical data
  • Glucokinase / genetics
  • Hepatocyte Nuclear Factor 4 / genetics
  • Humans
  • Male
  • Markov Chains
  • Maternal Inheritance*
  • Monte Carlo Method
  • Polymerase Chain Reaction / methods
  • Polymerase Chain Reaction / statistics & numerical data
  • Pregnancy
  • Prenatal Diagnosis / methods*


  • Biomarkers
  • HNF4A protein, human
  • Hepatocyte Nuclear Factor 4
  • DNA
  • Glucokinase