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Multicenter Study
. 2011 Jan 11:342:c7401.
doi: 10.1136/bmj.c7401.

Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study

Rossa W K Chiu  1 , Ranjit Akolekar, Yama W L Zheng, Tak Y Leung, Hao Sun, K C Allen Chan, Fiona M F Lun, Attie T J I Go, Elizabeth T Lau, William W K To, Wing C Leung, Rebecca Y K Tang, Sidney K C Au-Yeung, Helena Lam, Yu Y Kung, Xiuqing Zhang, John M G van Vugt, Ryoko Minekawa, Mary H Y Tang, Jun Wang, Cees B M Oudejans, Tze K Lau, Kypros H Nicolaides, Y M Dennis Lo
Affiliations
Multicenter Study

Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study

Rossa W K Chiu et al. BMJ. .

Abstract

Objectives: To validate the clinical efficacy and practical feasibility of massively parallel maternal plasma DNA sequencing to screen for fetal trisomy 21 among high risk pregnancies clinically indicated for amniocentesis or chorionic villus sampling.

Design: Diagnostic accuracy validated against full karyotyping, using prospectively collected or archived maternal plasma samples.

Setting: Prenatal diagnostic units in Hong Kong, United Kingdom, and the Netherlands.

Participants: 753 pregnant women at high risk for fetal trisomy 21 who underwent definitive diagnosis by full karyotyping, of whom 86 had a fetus with trisomy 21. Intervention Multiplexed massively parallel sequencing of DNA molecules in maternal plasma according to two protocols with different levels of sample throughput: 2-plex and 8-plex sequencing.

Main outcome measures: Proportion of DNA molecules that originated from chromosome 21. A trisomy 21 fetus was diagnosed when the z score for the proportion of chromosome 21 DNA molecules was >3. Diagnostic sensitivity, specificity, positive predictive value, and negative predictive value were calculated for trisomy 21 detection.

Results: Results were available from 753 pregnancies with the 8-plex sequencing protocol and from 314 pregnancies with the 2-plex protocol. The performance of the 2-plex protocol was superior to that of the 8-plex protocol. With the 2-plex protocol, trisomy 21 fetuses were detected at 100% sensitivity and 97.9% specificity, which resulted in a positive predictive value of 96.6% and negative predictive value of 100%. The 8-plex protocol detected 79.1% of the trisomy 21 fetuses and 98.9% specificity, giving a positive predictive value of 91.9% and negative predictive value of 96.9%.

Conclusion: Multiplexed maternal plasma DNA sequencing analysis could be used to rule out fetal trisomy 21 among high risk pregnancies. If referrals for amniocentesis or chorionic villus sampling were based on the sequencing test results, about 98% of the invasive diagnostic procedures could be avoided.

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Conflict of interest statement

Competing interests: All authors have completed the Unified Competing Interest form at www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare that (1) RWKC, HS, ETL, MHYT, TKL, and YMDL had support from the University Grants Committee of the Government of the Hong Kong Special Administrative Region, China, under the Areas of Excellence Scheme (AoE/M-04/06) for the submitted work; RWKC and YMDL had support from Sequenom for the submitted work; YMDL has support from an endowed chair from the Li Ka Shing Foundation. (2) YMDL is a consultant to, and holds equities in, Sequenom; RWKC and YMDL have received travel grants from Life Technologies and Illumina; RWKC, YWLZ, HS, KCAC, FMFL, and YMDL have received travel grants from University Grants Committee of the Government of the Hong Kong Special Administrative Region, China, under the Areas of Excellence Scheme (AoE/M-04/06). (3) The authors’ spouses, partners, and children have no financial relationships that may be relevant to the submitted work. (4) RWKC, YWLZ, KCAC, FMFL, and YMDL have filed patent applications on the detection of fetal nucleic acids in maternal plasma for non-invasive prenatal diagnosis. Part of this patent portfolio has been licensed to Sequenom and the Institut Jacques Boyd. The funders and sponsors have no role in the study design and the collection, analysis, and interpretation of data and the writing of the article and the decision to submit it for publication. The researchers are independent of the funders and sponsors.

Figures

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Fig 1 Recruitment of participants. Numbers in parentheses are the number of cases with sequencing results from the 2-plex protocol
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Fig 2 Z scores of percentage chromosome 21 (proportion of sequenced plasma DNA molecules originating from chromosome 21) determined by the 8-plex and 2-plex sequencing protocols. Broken lines indicate the z score cut-off value of 3
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Fig 3 Diagnostic efficacy of diagnosis of fetal trisomy 21 by sequencing of maternal plasma DNA according to 8-plex and 2-plex protocols. Receiver operating characteristic (ROC) curves (faint lines showing 95% confidence intervals) for measurements of percentage chromosome 21
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Fig 4 Fetal DNA concentration found in maternal plasma among pregnancies with euploid male fetuses
See this image and copyright information in PMC

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