High-throughput fetal fraction amplification increases analytical performance of noninvasive prenatal screening

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Abstract

Purpose

The percentage of a maternal cell-free DNA (cfDNA) sample that is fetal-derived (the fetal fraction; FF) is a key driver of the sensitivity and specificity of noninvasive prenatal screening (NIPS). On certain NIPS platforms, >20% of women with high body mass index (and >5% overall) receive a test failure due to low FF (<4%).

Methods

A scalable fetal fraction amplification (FFA) technology was analytically validated on 1264 samples undergoing whole-genome sequencing (WGS)–based NIPS. All samples were tested with and without FFA.

Results

Zero samples had FF < 4% when screened with FFA, whereas 1 in 25 of these same patients had FF < 4% without FFA. The average increase in FF was 3.9-fold for samples with low FF (2.3-fold overall) and 99.8% had higher FF with FFA. For all abnormalities screened on NIPS, z-scores increased 2.2-fold on average in positive samples and remained unchanged in negative samples, powering an increase in NIPS sensitivity and specificity.

Conclusion

FFA transforms low-FF samples into high-FF samples. By combining FFA with WGS–based NIPS, a single round of NIPS can provide nearly all women with confident results about the broad range of potential fetal chromosomal abnormalities across the genome.

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Most cited references 43

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Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood.

H Christina Fan, Yair Blumenfeld, Usha Chitkara … (2008)

We directly sequenced cell-free DNA with high-throughput shotgun sequencing technology from plasma of pregnant women, obtaining, on average, 5 million sequence tags per patient sample. This enabled us to measure the over- and underrepresentation of chromosomes from an aneuploid fetus. The sequencing approach is polymorphism-independent and therefore universally applicable for the noninvasive detection of fetal aneuploidy. Using this method, we successfully identified all nine cases of trisomy 21 (Down syndrome), two cases of trisomy 18 (Edward syndrome), and one case of trisomy 13 (Patau syndrome) in a cohort of 18 normal and aneuploid pregnancies; trisomy was detected at gestational ages as early as the 14th week. Direct sequencing also allowed us to study the characteristics of cell-free plasma DNA, and we found evidence that this DNA is enriched for sequences from nucleosomes.

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Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments.

Kun Sun, Peiyong Jiang, K. C. Allen Chan … (2015)

Plasma consists of DNA released from multiple tissues within the body. Using genome-wide bisulfite sequencing of plasma DNA and deconvolution of the sequencing data with reference to methylation profiles of different tissues, we developed a general approach for studying the major tissue contributors to the circulating DNA pool. We tested this method in pregnant women, patients with hepatocellular carcinoma, and subjects following bone marrow and liver transplantation. In most subjects, white blood cells were the predominant contributors to the circulating DNA pool. The placental contributions in the plasma of pregnant women correlated with the proportional contributions as revealed by fetal-specific genetic markers. The graft-derived contributions to the plasma in the transplant recipients correlated with those determined using donor-specific genetic markers. Patients with hepatocellular carcinoma showed elevated plasma DNA contributions from the liver, which correlated with measurements made using tumor-associated copy number aberrations. In hepatocellular carcinoma patients and in pregnant women exhibiting copy number aberrations in plasma, comparison of methylation deconvolution results using genomic regions with different copy number status pinpointed the tissue type responsible for the aberrations. In a pregnant woman diagnosed as having follicular lymphoma during pregnancy, methylation deconvolution indicated a grossly elevated contribution from B cells into the plasma DNA pool and localized B cells as the origin of the copy number aberrations observed in plasma. This method may serve as a powerful tool for assessing a wide range of physiological and pathological conditions based on the identification of perturbed proportional contributions of different tissues into plasma.

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DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation study.

Glenn E. Palomaki, Edward Kloza, Geralyn M Lambert-Messerlian … (2011)

Prenatal screening for Down syndrome has improved, but the number of resulting invasive diagnostic procedures remains problematic. Measurement of circulating cell-free DNA in maternal plasma might offer improvement. A blinded, nested case-control study was designed within a cohort of 4664 pregnancies at high risk for Down syndrome. Fetal karyotyping was compared with an internally validated, laboratory-developed test based on next-generation sequencing in 212 Down syndrome and 1484 matched euploid pregnancies. None had been previously tested. Primary testing occurred at a CLIA-certified commercial laboratory, with cross validation by a CLIA-certified university laboratory. Down syndrome detection rate was 98.6% (209/212), the false-positive rate was 0.20% (3/1471), and the testing failed in 13 pregnancies (0.8%); all were euploid. Before unblinding, the primary testing laboratory also reported multiple alternative interpretations. Adjusting chromosome 21 counts for guanine cytosine base content had the largest impact on improving performance. When applied to high-risk pregnancies, measuring maternal plasma DNA detects nearly all cases of Down syndrome at a very low false-positive rate. This method can substantially reduce the need for invasive diagnostic procedures and attendant procedure-related fetal losses. Although implementation issues need to be addressed, the evidence supports introducing this testing on a clinical basis.

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Author and article information

Contributors

Dale Muzzey:

ORCID: http://orcid.org/0000-0001-8822-2035

mwh_research@myriad.com

Journal

Journal ID (nlm-ta): Genet Med

Journal ID (iso-abbrev): Genet Med

Title: Genetics in Medicine

Publisher: Nature Publishing Group US (New York )

ISSN (Print): 1098-3600

ISSN (Electronic): 1530-0366

Publication date (Electronic): 15 November 2020

Publication date PMC-release: 15 November 2020

Publication date (Print): 2021

Volume: 23

Issue: 3

Pages: 443-450

Affiliations

[1 ]GRID grid.420032.7, ISNI 0000 0004 0460 790X, Myriad Genetics, Inc., ; Salt Lake City, UT USA

[2 ]Myriad Women’s Health, South San Francisco, CA USA

Author information

Dale Muzzey http://orcid.org/0000-0001-8822-2035

Article

Publisher ID: 1009

DOI: 10.1038/s41436-020-01009-5

PMC ID: 7935715

PubMed ID: 33190143

SO-VID: c50c0618-1261-40d7-986b-812f96f1733d

License:

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

History

Date received : 30 June 2020

Date revision received : 1 October 2020

Date accepted : 6 October 2020

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ScienceOpen disciplines: Genetics

Keywords: fetal fraction,cell-free dna,noninvasive prenatal screening,analytical validation,body mass index

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ScienceOpen disciplines: Genetics

Keywords: fetal fraction, cell-free dna, noninvasive prenatal screening, analytical validation, body mass index

High-throughput fetal fraction amplification increases analytical performance of noninvasive prenatal screening

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Abstract

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Conclusion

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Karger: Nutrition and Dietetics

Most cited references 43

Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood.

Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments.

DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation study.

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