Abstract
BackgroundBabies 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 4a (HNF4A) diabetes variant are at increased risk of high birth weight. Noninvasive fetal genotyping for maternal pathogenic variants would inform pregnancy management.MethodsDroplet 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.ResultsDroplet digital PCR assays for GCKor 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 falsepositive results. In 30 of 33 pregnancies, a result was obtained from a single blood sample.ConclusionsThis assay can be used to identify pregnancies at risk of macrosomia due to maternal monogenic diabetes variants.
Highlights
Prenatal diagnostic testing has been transformed through the discovery in 1997 of cell-free fetal DNA in maternal plasma during pregnancy [1]
The cellfree DNA (cfDNA) samples were assayed by droplet digital PCR (ddPCR) to quantify alleles of the maternal GCK or hepatocyte nuclear factor 4a (HNF4A) variant and the informative SNP identified for determination of fetal fraction (Supplemental Table 1)
At the lower range of fetal fraction, there was considerable overlap between data from heterozygous and homozygous pregnancies; this prevented confident prediction of fetal genotype based on simple inspection of ddPCR data
Summary
Prenatal diagnostic testing has been transformed through the discovery in 1997 of cell-free fetal DNA (cffDNA) in maternal plasma during pregnancy [1]. NIPT for maternally inherited variants that cause autosomal dominant disorders presents a particular challenge because only a small proportion of the total cfDNA in maternal blood is derived from the fetus during early pregnancy [12]. In such cases, a highly sensitive and precise method is required to quantify the presence of both reference and variant alleles to distinguish between fetal inheritance of the maternal variant—whereby cfDNA will show a 50:50 allelic ratio due to both mother and fetus being heterozygous—and noninheritance, where the normal allele will be overrepresented in proportion to the amount of fetal DNA present within the sample. Noninvasive fetal genotyping for maternal pathogenic variants would inform pregnancy management
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