NEW YORK (GenomeWeb) – Over the last few years, noninvasive prenatal testing has been established for fetal chromosomal aneuploidy screening as well as to look for subchromosomal aberrations, such as microdeletions and microduplications. However, when the mother unwittingly carries a chromosomal or subchromosomal abnormality herself, many types of NIPT that rely on counting DNA molecules cannot tell whether the fetus has inherited it as well.
Now researchers from the Chinese University of Hong Kong and their Dutch collaborators have demonstrated that an approach that combines size differences between fetal and maternal cell-free DNA with a counting-based method can determine whether the mother, fetus, or both carry a suspected subchromosomal copy number aberration. The results of the proof-of-concept study were published in Clinical Chemistry last week.
According to Rossa Chiu, professor of chemical pathology at the Chinese University of Hong Kong, the study is a continuation of earlier work from her team that was published in the Proceedings of the National Academy of Sciences in 2014 and exploited size differences between fetal and maternal cell-free DNA for diagnostic purposes.
In that study, Chiu and her colleagues showed that the differences in DNA fragment size can be used to detect fetal trisomies 21, 18, and 13, and monosomy X. The current study extends the method to subchromosomal aberrations.
The approach helps to clarify the origin of a subchromosomal abnormality. If the count-based approach, which enumerates DNA molecules from different genomic regions and looks for their over- or underrepresentation, suggests that a mother has a subchromosomal abnormality, it is impossible to tell for sure whether the fetus has also inherited it because the amount of fetal DNA in maternal blood is masked by the much larger amount of maternal DNA.
The size-based method exploits that fetal DNA fragments in maternal plasma are slightly smaller on average than maternal DNA fragments. An algorithm can compare the amount of short DNA fragments between genomic regions and determine whether there is a statistically significant excess or lack of those fragments. If both the mother and the fetus have the same abnormality, the fragment size distribution in that area would not change, but if only the mother has it and the fetus does not, or vice versa, the size distribution would change.
Unlike a count-based method, which can be performed with single-end fragment sequencing, the size-based approach requires paired-end sequencing, making it slightly more expensive, Chiu said.
But the sizing method also provides an independent measure of fetal DNA fraction in the maternal plasma sample, which Chiu said is an important quality control parameter in NIPT.
For their latest study, the researchers selected 10 cases with confirmed copy number aberrations in the fetus, the mother, or both. They performed paired-end sequencing on maternal plasma DNA from these cases, using the Illumina HiSeq platform, and performed count-based and size-based analyses. In all cases, the assay was able to correctly determine the origin of the subchromosomal abnormality.
In their paper, the researchers suggest that the approach could be used to lower the false positive rate of NIPT for fetal subchromosomal aberrations because the size-based analysis provides an independent measure. This, in turn, could lower the number of confirmatory invasive procedures required to follow up on false-positive results. The researchers have filed several US patent applications related to different steps of the method, Chiu said.
Chiu's lab has already implemented the size-based method routinely for all NIPT to cross-validate results from the count-based method and has applied it to thousands of cases. For aneuploidy testing, "although there is a low chance for a false positive [to occur from the counting method], we want to screen out those occasional cases," she said.
The lab performs paired-end sequencing for all NIPT, so no additional sequencing was needed to implement the size-based analysis. Comparing the results with the actual pregnancy outcomes will now allow the researchers to determine how accurate the size-based method is. "We're waiting to do the formal analysis of the accuracy in the thousands of cases," she said.
At least one other lab, Labco Diagnostics in Barcelona, Spain, has implemented a similar paired-end sequencing-based NIPT. Last week, that group published a performance evaluation of its test, called NeoBona, in Ultrasound in Obstetrics and Gynecology, for which it analyzed 1,000 samples from women undergoing screening for fetal trisomies 21, 18, and 13 and found that it could provide accurate results even for samples with low fetal DNA fractions.
Chiu said the main application of the size-based approach will likely be for assessing sex chromososomal aneuploidies. These types of aneuploidies, which include monosomy X, or Turner's syndrome, are relatively frequent, she said, and a surprising number of pregnant women — 8.6 percent of those who had a positive NIPT for a sex chromosomal aneuploidy in one study — have the chromosomal defect but are not aware of it.
In those cases, she said, a size-based NIPT can suggest right away whether the aneuploidy resides with the mother and whether she has likely passed it on to the fetus. In both cases, the lab currently recommends confirmatory testing, but even the suspicion that the mother has a chromosomal aneuploidy herself is "an important piece of information for counseling of future pregnancies," she said. "I think that is the biggest clinical need that this protocol is currently fulfilling in my routine practice."