NEW YORK (GenomeWeb) – Noninvasive prenatal testing company NIPD Genetics plans to add testing for subchromosomal alterations to its Veracity NIPT later this year. The firm recently published a proof-of-concept study in PLOS One, demonstrating that in artificial samples, its targeted sequencing approach can detect seven subchromosomal alterations.
Philippos Patsalis, CEO of the Cyprus-based company, said in an interview that NIPD Genetics has since developed an upgraded version of the test and will validate it in both artificial and clinical samples before making it available at its CAP accredited laboratory in Nicosia by the end of March.
The company launched Veracity, which is based on a proprietary targeted sequencing and analysis approach, in 2015, and the test is now available in 12 countries, Patsalis said. Sales have grown 300 percent since its launch, he said, and the firm raised €5 million ($5.4 million) in a private financing round last year.
In order to detect subchromosomal alterations, the company harnessed its existing targeted sequencing approach. The firm uses what it calls TACS for targeted capture sequences, which are essentially baits that it develops in house to target certain sequences. The TACS are designed to target genomic regions with certain characteristics: no known copy number variants, not in or near repetitive elements, and not in areas with complex 3D structures. The TACS then enrich the selected genomic regions through in-solution hybridization of an already prepared Illumina sequencing library. In this study, the team designed 755 TACS to analyze seven syndromes: 1p36 deletion, 22q11.2 deletion, Smith-Magenis, Potocki-Lupski, Miller-Dieker, NF1 microdeletion, and Wolf-Hirschhorn. In addition, 490 TACS were used to target reference regions on chromosomes 1 and 12.
Patsalis said that the group has since upgraded the test to include more TACS that take into consideration the complexity of the human genome in those regions.
For the proof-of-concept study, the NIPD Genetics team looked at artificial samples that mimic affected pregnancies — they spiked DNA from affected samples acquired from the Cyprus Institute of Neurology and Genetics biobank into plasma DNA of non-pregnant women. As controls, the team analyzed samples spiked with normal genomic DNA and cell-free DNA samples from normal pregnancies. The researchers also sheared the DNA to an average of 200 base pairs in order to mimic cell-free DNA, which is fragmented.
For the spiked samples, they simulated various fetal fractions by spiking in the affected DNA at three different concentrations: 20 percent, 10 percent, and 5 percent.
Overall, they evaluated 33 normal pregnancies, 21 artificially created affected samples, and 17 artificially created unaffected samples. Two artificially generated affected samples were excluded from analysis because of low sequencing coverage. Of the remaining 19 affected samples, all were called with no false negatives. In addition, all unaffected samples were called correctly with no false positives.
Dennis Lo, director of the Li Ka Shing Institute of Health Sciences at the Chinese University of Hong Kong who developed the NIPT technology that was commercialized by Sequenom, said in an email that the assay would need to be analyzed in "a cohort of real cases of sufficient numbers" before conclusions could be drawn as to how well it performs. "There are many biophysical differences between the DNA carrying fetal genetic information that is released by the placenta into maternal plasma and artificial samples," he said.
For instance, he said, in order to simulate cell-free DNA, the NIPD Genetics group fragmented the DNA to an average of 200 bases. In reality, however, fetal cell-free DNA tends to be shorter than maternal cell-free DNA, with the most prevalent size being 140 bases, versus 166 bases for maternal DNA. In addition, recent work performed by Lo's group has found that fragmentation may not be random. Non-random fragmentation "could result in differences in the relative concentrations of different [genomic] regions in plasma," which is not captured in the artificial samples, he said.
Patsalis noted that NIPD Genetics will validate its test in clinical samples, and said that a clinical study will be completed by mid March. He added that an advantage of the targeted sequencing approach is that it enables the detection of smaller alterations, since samples can be sequenced to a higher depth. In the PLOS One study, the assay detected a microdeletion as small as 500 kilobases.
In addition, Patsalis said, the company is looking to demonstrate that it can detect other fetal abnormalities, like single-gene disorders. As a commercial test, customers will have three different options for Veracity. The test can be ordered to screen for trisomies 21, 18, and 13 only, or customers can add sex chromosome aneuploidy screening, the subchromosomal alteration panel, or both. Patsalis declined to disclose the prices of each version of the test and said that it varied by country.
He added that the firm also plans to expand into other areas of reproductive health — including preimplantation testing and postnatal testing — as well as oncology. Its work with cell-free DNA is relevant not only for noninvasive prenatal testing, Patsalis said, but also in the cancer liquid biopsy space. The company has also been continuing its epigenetic research of "identifying and characterizing methylation-based markers that can potentially be used for prenatal testing," he said.