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

Author:

Caswell Richard C12,Snowsill Tristan3,Houghton Jayne A L12,Chakera Ali J14,Shepherd Maggie H15,Laver Thomas W1,Knight Bridget A15,Wright David6,Hattersley Andrew T12,Ellard Sian12

Affiliation:

1. Institute of Biomedical and Clinical Science, University of Exeter Medical School, Exeter, UK

2. Exeter Genomics Laboratory, Royal Devon and Exeter NHS Foundation Trust, Exeter, UK

3. Institute of Health Research, University of Exeter Medical School, Exeter, UK

4. Royal Sussex County Hospital, Brighton and Sussex University Hospitals NHS Trust, Brighton, UK

5. Exeter NIHR Clinical Research Facility, Royal Devon and Exeter NHS Foundation Trust, Exeter, UK

6. Institute of Health Research, University of Exeter Medical School, Knowledge Spa, Royal Cornwall Hospital, Truro, UK

Abstract

Abstract 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.

Funder

National Institute for Health Research

NIHR

University of Exeter Medical School College of Medicine and Health

Royal Devon and Exeter NHS Foundation Trust

Publisher

Oxford University Press (OUP)

Subject

Biochemistry, medical,Clinical Biochemistry

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