Researchers Eye Extending NIPT for Subchromosomal Analysis

Non-invasive prenatal testing (NIPT) is rapidly being adopted as an aneuploidy screening test in high-risk pregnancies. Its benefits include its high sensitivity and its ability to reduce the need for invasive testing. NIPT uses massively parallel sequencing of cell-free DNA in maternal plasma to detect fetal trisomies in chromosomes 13, 18, and 21.

While some are working towards evaluating the benefits of screening wider populations of women with NIPT, others are interested in expanding the scope of testing to include selected subchromosomal abnormalities. But, the evidence base remains limited for this use.

There are no serum-based tests that specifically screen for subchromosomal abnormalities. Individually, these rearrangements are rare, but combined have an estimated, combined incidence close to Down syndrome, researchers say. There are some concerns that widespread NIPT use could actually decrease detection of other pathogenic rearrangements—detectable with microarray analysis from invasively derived samples—given the declining numbers of invasive procedures performed.

A few previous studies demonstrated the potential of extending NIPT to detect fetal deletion and duplication syndromes, but questions remain about the fetal DNA fraction and the depth of sequencing needed for accurate results. Two recent studies provide additional data to fill this gap.

Rearrangements Less than 6 Mb Difficult to Detect
Standard NIPT can detect the majority of large chromosomal rearrangements (greater than 6 Mb), but negative results cannot rule out the possibility of smaller rearrangements, according to the conclusions of a study published in the Jan. 7 issue of the American Journal of Human Genetics. The authors say that given this possibility of undetectable smaller rearrangements, NIPT screening tests should not be expanded to include subchromosomal assessment.

This inability to detect small rearrangements is due to current practical limits in sequencing depth and reporting of fetal fraction—the proportion of circulating fetal DNA compared to maternal DNA.

The U.K. study sequenced 31 maternal blood samples collected from a cohort of women undergoing invasive procedures for clinical indications. Fetal conditions were determined by conventional karyotyping, fluorescence in situ hybridization, microarray, or molecular techniques. The included samples had fetal copy number variants (CNVs) ranging in size from less than 3 Mb to 42 Mb on different chromosomes. There were also three cases of unbalanced translocations.

The test samples were first sequenced at 12-plex (24 samples to a flow cell)—the same read depth as the group's standard in-house pipeline for aneuploidy NIPT. If no anomaly was detected, re-sequencing at a higher depth across multiple additional 1.5-plex runs (three samples per flow cell) was performed until the CNV was detected. The group developed a calling pipeline based on a segmentation algorithm for the detection of these rearrangements in maternal plasma.

The researchers found that standard NIPT methodology for aneuploidy can be used to detect the majority of pathogenic chromosomal rearrangements detectable by standard karyotyping. This was doable using a refined bioinformatics pipeline and no extra sequencing costs. Using 12-plex sequencing, 15 of 18 fetal subchromosomal abnormalities larger than 6 Mb were detected (sensitivity, 83 percent; specificity, 99.6 percent). With a read depth to 120 million reads, test sensitivity increased to 94 percent for CNVs larger than 6 Mb and to 93 percent for CNVs larger than 1.5 Mb.

However, for samples with CNVs less than 6 Mb, five of 13 were detected. Of these, three had duplications for which the mother was a carrier, but the current bioinformatics pipeline and standard depth of sequencing could not determine whether the fetus also carried the CNV. The analysis pipeline was also tested on a set of 534 maternal blood samples with no known chromosomal abnormalities that had been sequenced with at least four million reads. Three false positives in two samples resulted in a specificity of 99.6 percent.

"The lack of an independent method for determining fetal fraction, especially for female fetuses, leads to uncertainty in test sensitivity, which currently has implications for this technique's future as a clinical diagnostic test," write the authors led by Kitty Lo, Ph.D., from the University College London in the United Kingdom. "Given that a significant benefit of using NIPT to screen for aneuploidy is the increased safety secondary to the reduced need for invasive testing, extending NIPT to include screening for subchromosomal rearrangements stands to reverse some of this benefit whilst not offering comprehensive detection of pathogenic rearrangements. The costs and benefits of extending NIPT to this indication should be seriously considered prior to routine implementation."

Considering Fetal Fraction, Maternal Relocations
Fetal fraction is recognized to affect test sensitivity, and negative results cannot be considered conclusive without reporting of fetal fraction. A second study, published Nov. 24, 2015, in the Proceedings of the National Academy of Sciences, addressed both of these challenges and said that detection of fetal subchromosomal abnormalities is a "viable extension" of NIPT.

The China-based group investigated expanding NIPT using a semiconductor sequencing platform (SSP) in women carrying high-risk fetuses (fetal structural abnormalities detected on ultrasound). They analyzed plasma from 1,456 pregnant women undergoing an invasive diagnostic procedure (amniocentesis or chorionic villus sampling) and estimated fetal DNA concentration based on the size distribution of DNA fragments. Subchromosomal abnormalities were validated with array comparative genomic hybridization.

The group previously published a study showing the viability of SSP for NIPT and says that SSP offers the potential advantages of lower cost, faster sequencing, and a smaller machine footprint, making them easier to deploy in clinical laboratories. The fetal DNA concentration was determined from the proportion of Y-chromosome sequences in maternal plasma. In a training data set, the researchers observed a correlation between the fetal DNA fraction estimated by the Z score of Y chromosome and the reads ratio of two DNA fragment size regions: 130–140 basepairs (bp) and 155–175 bp. In the prospective cohort of 1,476 women, 66.7 percent of samples with a gestational age of 12 to 16 week had a fetal DNA fraction greater than 10 percent, compared with 95 percent of samples with gestational age greater than 20 weeks.

"Because the fetal concentration of DNA is lower at a gestational age of 12 to 16 weeks, … deeper sequencing than 3.5 million reads is likely required for accurate detection of subchromosomal abnormalities," write the authors led by Ai-hua Yin, from the Maternal and Children Metabolic-Genetic Key Laboratory at Guangdong Women and Children Hospital in China. "The current cost of sequencing a human genome with 0.5× coverage by single reads, approximately the coverage in our study at 3.5 million reads, is comparable with the cost of chromosomal microarray analysis while avoiding an invasive procedure. This depth allowed excellent detection of deletions/duplications greater than 5 Mb in size in our study. Deeper sequencing allows for finer resolution at an additional cost."

Several false positives in the full sample were due to low estimated fetal DNA fraction or samples in which a fraction could not be "reliably calculated." However, the majority of false positives (35 of 55) were due to deletions and/or duplications present in maternal DNA. This, the authors say, points to the necessity of reflex follow-up testing to rule out maternal DNA subchromosomal abnormalities, before a fetus can be diagnosed.

Takeaway: Despite eagerness to expand NIPT for screening for subchromosomal rearrangements, questions remain about the depth of sequencing and determination of fetal fraction needed to ensure accuracy of results.