Noninvasive Detection of Fetal Genetic Variations through Polymorphic Sites Sequencing of Maternal Plasma DNA


Noninvasive prenatal testing, often known as NIPT, is a procedure used to assess the likelihood that a fetus will be born with specific genetic disorders. Small DNA fragments that are floating about in a pregnant woman’s blood are examined by this test. These pieces are known as cell-free DNA because they are free-floating and not contained within cells, in contrast to most DNA, which is found inside the nucleus of a cell (cfDNA). When cells break down and die, their contents, including DNA, are released into the bloodstream as minute fragments with an average size of fewer than 200 DNA base pairs. The mother’s bloodstream during pregnancy contains a mixture of cfDNA from her cells and cells from the placenta. The tissue in the uterus known as the placenta connects the mother’s blood supply with the developing fetus. Throughout the pregnancy, these cells are released into the mother’s blood. Typically, the DNA of the placenta and the fetus are similar. Without endangering the fetus, cfDNA analysis from the placenta offers a chance for early diagnosis of some genetic disorders. Aneuploidy is characterized by the addition or deletion of one or more chromosomes. With rising maternal age, the risk of fetal aneuploidy increases. Pregnant women should have access to screening since fetal aneuploidy can damage every pregnancy.

Relative allelic counts can be used to identify fetal aneuploidies when the fetus gets a paternal allele that differs from the mother’s. Song Gao from Wuhan University recently published a new exciting study in The Journal of Gene Medicine where he proposed a novel assay, noninvasive prenatal testing based on goodness-of-fit and graphical analysis of polymorphic sites (GGAP-NIPT), to simultaneously detect fetal abnormalities at the chromosomal/subchromosomal and monogenic sequence levels. The assay was shown to be sensitive and accurate for all test samples.

The author described a method to simultaneously detect chromosomal, subchromosomal, and sequence-level abnormalities using amplicon sequencing of polymorphic and target sites of maternal plasma DNA. A panel of polymorphic sites from presumably healthy reference chromosomes, a panel of polymorphic sites on the target chromosomal/subchromosomal regions, and a panel of specific target sites from monogenic mutation hotspots were amplified, sequenced, and analyzed for each testee. Allelic read counts from each polymorphic site preserved the relative ratios among its different alleles, which formed the basis of the new assay to estimate fetal percentage and the genotypes of target sites. Allelic read counts on reference chromosomes were linearly processed to estimate the fetal percentage with a robust linear regression model fitting. This process kept linear correlations between variables while making the data easier to interpret and making the association between variables clear and impervious to outliers. Following fetal percentage estimation, the underlying genotype of each target site was assessed graphically and quantitatively. For three open datasets, over 90% of the target monogenic mutations were accurately identified, which could be further improved when samples were examined using replicates, whereas it is challenging to use the currently accessible tools to assess a group of replicated values.

Song Gao found the modified ∆AIC value to be a good indicator of how well allelic counts fit with the estimated genotype in the detection of target mutation using allelic counts of a single site, and groups of such sites could also be assessed collectively in a similar manner. For monogenic samples with replicates, groups of values for the same target were assessed altogether, and improved detection accuracy was achieved. For aneuploidies, microdeletions, and microduplications, groups of distinct target sites were collectively analyzed. As the same underlying strategy was applied to detect chromosomal/subchromosomal abnormalities and monogenic mutations, all genetic changes could be detected simultaneously, and no comparisons were made across samples or sites. In addition, such a detecting strategy could be applied to detect genetic changes in a surrogate mother’s cfDNA sample, whereas different genotype models were used to estimate the fetal fraction and determine the genotype of each target site.

In conclusion, Song Gao designed a novel assay called GGAP-NIPT for noninvasively identifying embryonic genetic disorders at the chromosomal/subchromosomal and nucleotide levels with proven accuracy for simulated samples. Such an assay demonstrated the potential to assist the expansion of NIPT to identify genetic illnesses with high prevalence in certain populations and conditions common to all pregnancies, which would have significant socioeconomic benefits. It is noteworthy to mention a patent has been filed to protect the new technology.


Gao S. Noninvasive detection of fetal genetic variations through polymorphic site sequencing of maternal plasma DNA. The Journal of Gene Medicine. 2022 Mar;24(3):e3400.

Go To The Journal of Gene Medicine.