Institute of Biomedical and Clinical Science, University of Exeter Medical School, Exeter, UK.
Exeter Genomics Laboratory, Royal Devon and Exeter NHS Foundation Trust, Exeter, UK.
Clin Chem. 2020 Jul 1;66(7):958-965. doi: 10.1093/clinchem/hvaa104.
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.
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.
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.
This assay can be used to identify pregnancies at risk of macrosomia due to maternal monogenic diabetes variants.
携带导致轻度空腹性高血糖的杂合致病性葡萄糖激酶（GCK）变异的女性所生婴儿，如果不遗传该变异，则有发生巨大儿的风险。相反，如果遗传了致病性肝细胞核因子 4α（HNF4A）糖尿病变异，则婴儿发生高出生体重的风险增加。对母体致病性变异进行非侵入性胎儿基因分型可以为妊娠管理提供信息。
使用液滴数字 PCR 技术定量分析 38 名携带 GCK 或 HNF4A 变异的杂合子孕妇血液中的游离 DNA 中的参考和变异等位基因，并通过测量有意义的母体和父体变异来确定胎儿分数。用阳性参考/等位基因的液滴数与胎儿分数一起用于贝叶斯分析，以得出胎儿基因型的概率。婴儿的基因型通过 Sanger 测序在产后确定。
针对 GCK 或 HNF4A 变异的液滴数字 PCR 检测方法在所有 38 例妊娠中均得到验证。在至少 1 份来自 33 例妊娠的游离 DNA 样本中证实了胎儿分数≥2%。对于纯合参考基因型的调用阈值≥0.95，杂合胎儿基因型的调用阈值≤0.05，允许对所有 33 例妊娠进行正确的基因型调用，且没有假阳性结果。在 33 例妊娠中的 30 例中，从单个血样中获得了结果。
该检测方法可用于识别因母体单基因糖尿病变异而有发生巨大儿风险的妊娠。
