Noninvasive Prenatal Screening of Fetal Aneuploidy without Massively Parallel Sequencing

Abstract BACKGROUND Noninvasive prenatal screening (NIPS) using plasma cell-free DNA has gained tremendous popularity in the clinical assessment of fetal aneuploidy. Most, if not all, of these tests rely on complex and expensive massively parallel sequencing (MPS) techniques, hindering the use of NIPS as a common screening procedure. METHODS We have developed and optimized an MPS-independent noninvasive genetic test that can rapidly detect fetal aneuploidy at considerably lower costs. We used the high-throughput ligation-dependent probe amplification (HLPA) assay with standard z score statistics to identify the minute copy number change of targeted chromosomal regions. HLPA was modified from multiplex ligation-dependent probe amplification to allow quantification of up to 200 genomic loci in a single multiplex PCR. As a proof of principle, we conducted Down syndrome screening in 1182 women with singleton pregnancies [maternal age (SD): 32.7 (4.6)] using whole-genome sequencing-based NIPS and our method. RESULTS Nineteen fetuses with trisomy 21 were detected by both methods and confirmed by karyotyping of amniotic fluid. Overall, our method showed 100.0% sensitivity (19/19) and 99.7% specificity (1076/1079) in trisomy 21 screening, generating a positive predictive value of 86.4% (19/22) and a 7.1% (84/1182) no-call rate. CONCLUSIONS Our technique potentially opens new avenues for the development of inexpensive, yet effective, prenatal aneuploidy tests. The simplicity and accuracy of this method make it a good candidate for clinical implementation as a standard screening procedure.

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Noninvasive Prenatal Screening for Genetic Diseases Using Massively Parallel Sequencing of Maternal Plasma DNA

Cold Spring Harbor Perspectives in Medicine ◽

10.1101/cshperspect.a023085 ◽

2015 ◽

Vol 5 (9) ◽

pp. a023085 ◽

Cited By ~ 36

Author(s):

Lyn S. Chitty ◽

Y. M. Dennis Lo

Keyword(s):

Prenatal Screening ◽

Massively Parallel Sequencing ◽

Genetic Diseases ◽

Maternal Plasma ◽

Massively Parallel ◽

Plasma Dna ◽

Parallel Sequencing ◽

Noninvasive Prenatal Screening

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External Quality Assessment for Detection of Fetal Trisomy 21, 18, and 13 by Massively Parallel Sequencing in Clinical Laboratories

Journal of Molecular Diagnostics ◽

10.1016/j.jmoldx.2015.10.003 ◽

2016 ◽

Vol 18 (2) ◽

pp. 244-252 ◽

Cited By ~ 5

Author(s):

Rui Zhang ◽

Hongyun Zhang ◽

Yulong Li ◽

Yanxi Han ◽

Jiehong Xie ◽

...

Keyword(s):

Quality Assessment ◽

Trisomy 21 ◽

External Quality Assessment ◽

Massively Parallel Sequencing ◽

Massively Parallel ◽

External Quality ◽

Parallel Sequencing ◽

Clinical Laboratories

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Non-invasive prenatal testing for fetal aneuploidy by massively parallel DNA sequencing of maternal plasma: the future has arrived today/Nicht-invasiver Pränatal-Test auf fetale Aneuploidie mittels massiv-paralleler DNA-Sequenzanalyse im mütterlichen Plasma: in der Zukunft angekommen

LaboratoriumsMedizin ◽

10.1515/labmed-2012-0029 ◽

2012 ◽

Vol 36 (5) ◽

Cited By ~ 2

Author(s):

Amy Swanson ◽

Christin Coffeen ◽

Amy J. Sehnert

Keyword(s):

Massively Parallel Sequencing ◽

Prenatal Testing ◽

The United States ◽

Maternal Blood ◽

Maternal Plasma ◽

Massively Parallel ◽

Clinical Implementation ◽

Maternal Cell ◽

Non Invasive ◽

Fetal Aneuploidy

AbstractAfter decades of research, non-invasive prenatal testing (NIPT) using maternal blood to determine fetal chromosome status has found its way from the research laboratory into clinical practice, triggering a long-awaited paradigm shift in prenatal care. A variety of methods using sequencing of maternal cell-free DNA (cfDNA) have now been studied, primarily demonstrating their ability to detect the most common fetal aneuploidy, trisomy 21 (T21). The focus of this article is on massively parallel sequencing (MPS) with optimized sequence tag mapping and chromosome quantification, which accurately detects T21 as well as multiple other aneuploidies across the genome. The power of this technique resides in its high precision and reduction of variation within and between sequencing runs. Using MPS, classification of aneuploidy status for a given sample can be reliably assigned from the genetic information alone without the need to factor in other maternal pre-test risk or other clinical variables. Performance of this method has been prospectively demonstrated in a rigorous, blinded, multi-center study in the United States. The findings suggest that MPS can be incorporated into existing prenatal screening algorithms to reduce unnecessary invasive procedures. This technology and key considerations for clinical implementation are discussed.

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Maternal Plasma DNA Analysis with Massively Parallel Sequencing by Ligation for Noninvasive Prenatal Diagnosis of Trisomy 21

Clinical Chemistry ◽

10.1373/clinchem.2009.136507 ◽

2010 ◽

Vol 56 (3) ◽

pp. 459-463 ◽

Cited By ~ 99

Author(s):

Rossa WK Chiu ◽

Hao Sun ◽

Ranjit Akolekar ◽

Christopher Clouser ◽

Clarence Lee ◽

...

Keyword(s):

Prenatal Diagnosis ◽

Trisomy 21 ◽

Massively Parallel Sequencing ◽

Maternal Plasma ◽

Chromosome 21 ◽

Massively Parallel ◽

Plasma Dna ◽

Parallel Sequencing ◽

Noninvasive Prenatal Diagnosis ◽

Sequencing By Synthesis

Abstract Background: Noninvasive prenatal diagnosis of trisomy 21 (T21) has recently been shown to be achievable by massively parallel sequencing of maternal plasma on a sequencing-by-synthesis platform. The quantification of several other human chromosomes, including chromosomes 18 and 13, has been shown to be less precise, however, with quantitative biases related to the chromosomal GC content. Methods: Maternal plasma DNA from 10 euploid and 5 T21 pregnancies was sequenced with a sequencing-by-ligation approach. We calculated the genomic representations (GRs) of sequenced reads from each chromosome and their associated measurement CVs and compared the GRs of chromosome 21 (chr21) for the euploid and T21 pregnancies. Results: We obtained a median of 12 × 106 unique reads (21% of the total reads) per sample. The GRs deviated from those expected for some chromosomes but in a manner different from that previously reported for the sequencing-by-synthesis approach. Measurements of the GRs for chromosomes 18 and 13 were less precise than for chr21. z Scores of the GR of chr21 were increased in the T21 pregnancies, compared with the euploid pregnancies. Conclusions: Massively parallel sequencing-by-ligation of maternal plasma DNA was effective in identifying T21 fetuses noninvasively. The quantitative biases observed among the GRs of certain chromosomes were more likely based on analytical factors than biological factors. Further research is needed to enhance the precision for measuring for the representations of chromosomes 18 and 13.

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