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Fetal Exome Sequencing Studies Point to Noninvasive Prenatal Screening Applications

NEW YORK – In a pair of papers published in the New England Journal of Medicine last week, independent research teams have demonstrated the potential for using deep trio exome sequencing on parental samples and cell-free DNA (cfDNA) from maternal blood plasma for noninvasive prenatal screening testing to find pathogenic de novo or paternally inherited variants.

For the first of the studies, researchers from the Broad Institute, Massachusetts General Hospital, Brigham and Women's Hospital, and Boston Children's Hospital analyzed peripheral blood samples from 51 women in their first, second, or third trimester of pregnancy, extracting a mix of maternal and fetal cell-free DNA from the plasma.

They then sequenced the exome of the cell-free DNA with an average depth of 210-fold and identified the fetal portion of the exome with the help of paternal allele profiles generated in the parental exome sequencing step.

"Gestational ages spanned the trimesters, with [five] pregnancies in the first trimester, [nine] in the second, and 37 in the third," corresponding author Michael Talkowski, director of Mass General Hospital's Center for Genomic Medicine, and his colleagues wrote, noting that the proportion of cfDNA from the fetus or placenta ranged from 6 percent up to 51 percent.

"These results suggest that high-resolution noninvasive prenatal screening may provide a robust fetal screen at nucleotide resolution for de novo variants across gestational ages, with the potential to detect maternally inherited and recessive disease variants at fetal fractions of more than 15 to 20 percent," the authors explained, noting that their proof-of-principle analyses "suggest that a fetal exome screen is accessible from maternal plasma samples that are already routinely obtained for fetal aneuploidy testing."

Along with a rising fetal DNA fraction in third trimester samples relative to second trimester or first trimester samples, the team successfully tracked down fetal and maternal variants in the cfDNA, using the exome-derived genotypes to perform prenatal screening for conditions such as Stickler syndrome and carrier screening for conditions such as phenylketonuria (PKU).

"The clinical implications of this research are potentially profound, particularly for pregnancies in which a fetal anomaly is suspected from ultrasound and an invasive test is indicated," coauthor Kathryn Gray, a researcher at Brigham and Women's Hospital, said in a statement.

"In instances where a current standard-of-care test identifies an abnormality during prenatal diagnostic testing, we ensure that patients have access to a multidisciplinary team, including maternal-fetal medicine and pediatric specialists, genetic counselors, and social workers, to help patients understand complicated test results," Gray explained. "Noninvasive tests, including currently available [noninvasive prenatal testing] screening methods, will require the same support network."

In another article in NEJM, investigators at the University of Southern Denmark, Odense University Hospital, and other Danish centers presented their own noninvasive prenatal screening strategy based on deep exome sequencing on parent-fetal trios.

Using cell-free DNA isolated from plasma samples from 36 pregnant women, members of that team performed exome sequencing at 2,710- to 8,075-fold coverage. The group went on to analyze the sequences with a custom pipeline spanning variant calling, error correction, and fetal fraction estimates, which came in at 3.72 percent to 19 percent in the cases considered.

"This proof-of-concept approach leverages ultra-deep, error-corrected, trio-exome sequencing that enables the detection of fetal de novo variants with high accuracy," the authors wrote, noting that their strategy "has high sensitivity for detecting fetal single-nucleotide variants, small insertions and deletions, large copy-number variants, and chromosomal aneuploidies from cell-free DNA obtained from a maternal blood sample."

By incorporating exome sequences from both parents, the researchers teased out de novo and paternally inherited variants in each fetus, flagging a handful of pathogenic fetal de novo variants in 11 pregnancies that were all validated using fetal samples. The noninvasive exome sequencing method did not miss any pathogenic variants found by exome sequencing, whole-genome sequencing, or microarray profiling on chorionic villus or amniotic fluid samples.

"No additional diagnostic de novo variants were identified by means of invasive analysis in the 36 pregnancies," the authors reported. "Thus, we observed full concordance between noninvasive prenatal screening using deep trio-exome sequencing and invasive prenatal analysis, with 100 percent detection for pathogenic de novo variants."

With insights from paternally inherited variants in the fetal exomes, meanwhile, the team estimated that the cfDNA-based deep exome sequencing approach picked up fetal variants with an average sensitivity of more than 95 percent, depending on the depth of coverage and fetal fraction in the cfDNA sample.

"Our study showed that this [trio-exome sequencing] approach to testing can accurately determine the fetal inheritance of paternal variants in the event that the mother does not carry the same variant," the researchers reported, noting that invasive testing would be needed to confirm maternally inherited variants.

Based on their findings, the authors suggested that "integration of noninvasive prenatal screening with deep trio-exome sequencing into routine prenatal care in conjunction with fetal ultrasonographic screening would provide an opportunity to improve early detection rates, reduce the number of invasive procedures, and facilitate prompt interventions."

"Although large studies are needed to test this hypothesis, results from our study … would predict a low false-positive rate," they explained, adding that "positive findings should be confirmed through prenatal invasive diagnostic testing."