NEW YORK (GenomeWeb News) – Massively parallel sequencing of cell-free DNA gathered from maternal blood samples can give researchers a view of fetal subchromosomal abnormalities that is equivalent to what would be obtained from a chromosomal microarray or metaphase karyotype, Verinata Health researchers reported today in the American Journal of Human Genetics.
The researchers, led by Richard Rava, the chief scientific officer of Verinata, examined maternal blood samples taken from pregnant women carrying fetuses with known subchromosomal duplications, deletions, and other changes to determine the accuracy of massively parallel sequencing for noninvasive prenatal testing.
Verinata offers Verifi, a prenatal blood test based on technology developed by Stephen Quake's lab at Stanford University that currently focuses on detecting trisomies and sex chromosome abnormalities. Earlier this week, Illumina announced that it plans to acquire Verinata for up to $450 million and that it will be submitting the Verifi test to the US Food and Drug Administration as an in vitro diagnostic test.
In the AJHG study, the Verinata researchers showed that deep sequencing of cell-free fetal DNA from maternal blood may be a viable addition to, or replacement for, analysis of fetal samples using karyotyping or chromosomal microarrays. "Our study is the first to show that almost all the information that is available from an invasive procedure is also available noninvasively from a simple maternal blood draw," Verinata's Rava and the study's senior author said in a statement.
While a number of sequencing-based tests — such as Verifi, Sequenom's MaterniT21, and Ariosa's Harmony — analyze maternal blood to detect whole-chromosomal aneuploidies, the Verinata team sought to show that the approach could be used to detect subchromosomal abnormalities as well.
How well massively parallel sequencing can detect fetal chromosomal abnormalities from maternal blood depends on the percentage of fetal DNA found in maternal blood samples — the higher the fraction, the better the approach should work.
Rava and his colleagues artificially combined fetal and maternal DNA at different fractions of fetal DNA — both 5 percent and 10 percent mixtures — to test how deep they'd have to sequence them to get a good look at the fetal DNA. Those samples, obtained from the Coriell Institute for Medical Research, represented a range of chromosomal abnormalities, from aneuploidies to mosaic sub-chromosomal copy-number changes.
Additionally, the researchers turned to 11 blood plasma samples from the MELISSA trial, which examined aneuplodies and other chromosomal aberrations in high-risk pregnant women, to test the approach on clinical samples.
Both sets of samples were tagged with 25-mer tags, with about a billion tags per sample, and sequenced with single-ended, 36-basepair reads on Illumina's HiSeq 2000 instrument. The reads were then mapped to the human reference and examined and classified for chromosomal abnormalities.
"This study demonstrates that in nonmosaic cases, it is possible to obtain a fetal molecular karyotype that is equivalent to CMA by MPS of maternal plasma cfDNA," the researchers wrote. "In some cases the MPS results provided better resolution than a metaphase karyotype obtained from chorionic villi or amniocytes."
Additionally, the investigators noted that their approach was able to identify where additional material found on chromosomes in two different samples may have originated from by detecting small deletions at likely breakpoints.
However, the inability to provide results on clinical mosaic samples points out the approach's limitations: Certain genomic regions close to the telomeres or centromeres remain inaccessible.
Despite the limitations, the researchers said that sequencing of cell-free fetal DNA from maternal blood to look for chromosomal abnormalities may be a stepping stone for introducing other prenatal diagnoses. Such an approach would likely also be safer as a blood draw eliminates the low, but possible, risk of miscarriage associated with amniocentesis and chorionic villus sampling.
"Although this work suggests an exciting future path toward routine noninvasive detection of the entire fetal genome, most clinicians are not yet ready to interpret the massive amounts of information that will come from the entire sequence," Rava and his colleagues wrote. "They are, however, already familiar with CMAs, so our work can potentially be translated to clinical care more expeditiously and eventually provide a rationale for whole-genome sequencing of the fetus."
The American College of Medical Genetics and Genomics updated its guidelines several years ago to recommend chromosomal microarrays as a first-tier diagnostic test for the postnatal evaluation of patients with developmental delay or intellectual disability, autism spectrum disorders, or multiple congenital anomalies. However, ACMG's guidelines continue to state that arrays should only be used as an adjunctive test to karyotyping in a prenatal setting.
The organization does not support the use of prenatal sequencing except for cases in which a fetus has "a likely genetic disorder in which specific genetic tests , including targeted sequencing tests, available for that phenotype have failed to arrive at a diagnosis," according to a policy statement issued last May.
ACMG notes in the statement that prenatal diagnosis by genomic sequencing has "significant limitations." Namely, "the current technology does not support short turn-around times which are often expected in the prenatal setting. There are high false positive, false negative, and variants of unknown clinical significance rates. These can be expected to be significantly higher than seen when array CGH is used in prenatal diagnosis."