Whole genome sequencing of cell-free DNA for NIPT


An analysis of a large cohort of patients who chose noninvasive prenatal testing (NIPT) as a screening method for fetal trisomy 21, 18, and 13 (T21, T18 and T13) and sex chromosome aneuploidies (SCA) concluded there were extremely high detection rates and exceptionally low false positive rates.

The retrospective study in the journal BMC Medical Genomics evaluated the performance of a whole genome sequencing (WGS)-based NIPT test in a general Italian pregnancy population in 35,650 singleton, 800 twin, and 6 triplet pregnancies.

Among the cohort, 46% had an a-priori elevated risk and 54% an a-priori low risk.

Samples were tested at the AMES Group laboratory in Naples, Italy, between April 2017 and September 2019.

“We wanted to reflect on the results obtained in our clinical laboratory and the outcomes of pregnancies tested, in order to improve the health of pregnant patients,” said principal investigator Luigia De Falco, PhD, a molecular biologist with a doctorate in molecular genetics, who is employed at the laboratory.

In 76.2% of patients, blood was drawn in the first trimester of pregnancy. The mean gestational age at the time of blood draw was 12 weeks and 2 days.

Maternal age ranged from 18 to 48 years (mean 35.4%). The average fetal cell-free DNA fraction was 9.54%.

In total, 5% of pregnancies were conceived via assisted reproductive technologies (ART) and 0.04% through egg donation.

Samples were sequenced on a NextSeq 550 (Illumina) using the VeriSeq NIPT Solution v1 assay.

Test results indicative of a classic trisomy were found in 1% (n = 356) of samples: T21 (n = 254), T18 (n = 69) and T13 (n = 33).

Test results indicative of a XCA were found in 0.4% (n = 145) of samples.

Of the combined 501 screen-positive cases, 484 had confirmatory diagnostic testing.

NIPT results were confirmed in 99.2% of T21 cases, 91.2% of T18 cases, 84.4% of T13 cases, and 86.7% of SCA cases.

Among the 35,955 cases reported as unaffected by a classic trisomy or SCA, no false negative cases were reported.

Assuming that false negative results would be reported, NIPT sensitivity was 100% for all 3 fetal trisomies: T21 (95% confidence interval [Cl]: 98.47 to 100.0), T18 (95% Cl: 94.17 to 100.0), and T13 (95% Cl: 87.54 to 100.0).

Similarly, the specificities were 99.99% (95% Cl: 99.98 to 100.0), 99.98% (95% Cl: 99.96 to 100.0), 99.99% (95% Cl: 99.97 to 100.0), and 99.95% (95% Cl: 99.92 to 99.97) for T21, T18, T13 and SCA, respectively.

“We were surprised by the extremely low failure rate of only 3 in 10,000 births by using our WGS NIPT,” De Falco told Contemporary OB/GYN®.

The investigators noted that WGS NIPT is reliable for pregnant women with multiple foetuses. “This is relevant, since in addition to the risk of an abnormal result, the risk of invasive test procedures is higher in twin pregnancies,” they wrote.

WGS-based NIPT estimates fetal fraction based on read lengths and coverage profiles. According to De Falco, it also allows for identifying an aneuploidy in a non-targeted chromosome, and avoids test failure due to such events.

These advantages, coupled with adherence to a strict decision tree for samples that fail at primary attempt, allow for highly accurate testing and an extremely low non-reporting rate for both singleton and multiple pregnancies.

“The study results will support obstetricians, midwives and geneticists in pretest and post-test counseling conversations,” De Falco said.

Cell-free DNA NIPT has “revolutionized prenatal screening for chromosome anomalies,” wrote the authors, and currently represents “the best-performing screening method for common autosomal aneuploidies for all pregnant women.”



De Falco reports no relevant financial disclosures.


La Verde M, De Falco L, Torella A, et al. Performance of cell-free DNA sequencing-based non-invasive prenatal testing: experience on 36,456 singleton and multiple pregnancies. BMC Med Genomics. Published online March 30, 2021. doi:10.1186/s12920-021-00941-y

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