RESEARCH
`
`Non-invasive prenatal assessment of trisomy 21 by
`multiplexed maternal plasma DNA sequencing: large scale
`validity study
`
`Rossa W K Chiu, professor,1 Ranjit Akolekar, clinical research fellow,3 Yama W L Zheng, student ,1 Tak Y Leung,
`professor,2 Hao Sun, assistant professor,1 K C Allen Chan, associate professor,1 Fiona M F Lun, postdoctoral
`fellow,1 Attie T J I Go, professor,4 Elizabeth T Lau, department manager and honorary assistant professor,5
`William W K To, consultant,6 Wing C Leung, consultant,7 Rebecca Y K Tang, consultant,8 Sidney K C Au-Yeung,
`consultant,9 Helena Lam, consultant,10 Yu Y Kung, obstetrician,11 Xiuqing Zhang, manager,12,13 John M G van
`Vugt, professor,4 Ryoko Minekawa, postdoctoral fellow,3 Mary H Y Tang, consultant and honorary clinical
`associate professor,5 Jun Wang, professor,12 associate director,13 Cees B M Oudejans, associate professor,4
`Tze K Lau, professor,2 Kypros H Nicolaides, professor,3 Y M Dennis Lo, professor1,12
`
`1Centre for Research into
`Circulating Fetal Nucleic Acids, Li
`Ka Shing Institute of Health
`Sciences, Department of Chemical
`Pathology, The Chinese University
`of Hong Kong, Hong Kong SAR,
`China
`2Department of Obstetrics and
`Gynaecology, The Chinese
`University of Hong Kong
`3Harris Birthright Research Centre
`for Foetal Medicine, King’s College
`Hospital, London SE5 9RS, UK
`4VU University Medical Center,
`10081 HV Amsterdam,
`Netherlands
`5Tsan Yuk Hospital, Department of
`Obstetrics and Gynaecology,
`University of Hong Kong, Hong
`Kong
`6United Christian Hospital,
`Hospital Authority, Hong Kong
`7Kwong Wah Hospital, Hospital
`Authority, Hong Kong
`8Pamela Youde Nethersole
`Eastern Hospital, Hospital
`Authority, Hong Kong
`9Tuen Mun Hospital, Hospital
`Authority, Hong Kong
`10Princess Margaret Hospital,
`Hospital Authority, Hong Kong
`11YY Kung Medical Centre, Hong
`Kong
`12Joint Chinese University of Hong
`Kong-Beijing Genomics Institute
`Genome Research Centre, Hong
`Kong
`13Beijing Genomics Institute at
`Shenzhen, Shenzhen, China
`Correspondence to: Y M D Lo
`loym@cuhk.edu.hk
`
`Cite this as: BMJ 2011;342:c7401
`doi:10.1136/bmj.c7401
`
`BMJ | ONLINE FIRST | bmj.com
`
`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.
`
`INTRODUCTION
`Trisomy 21, Down’s syndrome, occurs in 1 in 800 live
`births.1 Prenatal diagnosis of trisomy 21 requires inva-
`sive sampling of fetal genetic material through amnio-
`centesis or chorionic villus sampling. However, these
`tests carry a risk of miscarriage of about 1%,2 and they
`are therefore reserved for pregnancies considered to be
`at high risk of fetal trisomy 21. The traditional method
`of identifying the high risk group has been increased
`maternal age, but screening by this method would
`require invasive testing in about 5% of pregnant
`women and identify only 30% of affected fetuses.1 In
`the past 20 years maternal age has been combined with
`ultrasonographic examination of the fetus and bio-
`chemical measurement of various proteins or hor-
`mones
`in the maternal circulation to improve
`identification of high risk pregnancies. This combined
`approach of screening can now identify more than 90%
`of affected fetuses, but there is still a need for invasive
`testing in 3–5% of the population.3-9
`Cell-free DNA from the fetus has been found in the
`plasma of pregnant women, and this has been used
`successfully for non-invasive determination of the
`fetal sex and fetal RhD genotype in RhD negative
`women.10-13 The basis of these tests is the detection of
`fetal-specific DNA sequences in maternal plasma.14
`The same approach of searching for fetal-specific
`nucleic acids, such as DNA methylation and mRNA
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`markers in maternal plasma, has been proposed for
`non-invasive detection of fetal aneuploidies.15-19
`these
`The
`advantages
`and disadvantages of
`approaches have been reviewed in detail.20 An alterna-
`tive approach for non-invasive prenatal diagnosis of
`fetal trisomy 21 is to show the presence of an elevated
`amount of chromosome 21 sequences in maternal
`blood, because there are three rather than two copies
`of fetal chromosome 21.18 However, fetal DNA mole-
`cules amount to just 10–20% of the total DNA circulat-
`ing in the maternal plasma,21 22 so any increment in the
`total amount (fetal and maternal) of chromosome 21
`DNA molecules in the plasma of a trisomy 21 preg-
`nancy would be substantially diluted by contributions
`from the mother.20
`The difficulty in measuring such a small increment in
`chromosome 21 DNA concentration has recently been
`overcome with the use of massively parallel genomic
`sequencing.20 This technique can identify and quantify
`millions of DNA fragments in biological samples in a
`span of days.23 Three cohort studies have shown the
`feasibility of using the technique to identify fetuses
`with trisomy 21 by analysis of maternal plasma
`DNA.24-26 The sample numbers studied were small
`because, typically, only a few samples could be ana-
`lysed in each sequencing run.
`The objective of this study is to validate the diagnos-
`tic performance and practical feasibility of massively
`parallel genomic sequencing for the non-invasive pre-
`natal assessment of trisomy 21 in a large number of
`pregnancies that have undergone conventional screen-
`ing and were clinically indicated for definitive testing
`by amniocentesis or chorionic villus sampling.
`
`METHODS
`The primary research question of the study was to eval-
`uate if maternal plasma DNA sequencing could accu-
`rately confirm or exclude fetal trisomy 21 compared
`with full karyotyping among pregnancies clinically
`indicated for amniocentesis or chorionic villus sam-
`pling. A secondary goal of the study was to develop
`laboratory protocols to improve the throughput of
`massively parallel genomic sequencing for handling
`large sample numbers.
`
`Recruitment criteria
`We prospectively recruited pregnant women from
`eight obstetric units in Hong Kong, one unit from the
`Netherlands, and one prenatal diagnostic centre in the
`United Kingdom between October 2008 and May
`2009. We also retrieved archived maternal plasma
`samples collected between October 2003 and Septem-
`ber 2008 from trisomy 21 and non-trisomy 21 preg-
`nancies matched for gestational ages in a ratio of
`approximately 1:5 from the participating sites in the
`Netherlands and UK. The inclusion criteria were sin-
`gleton pregnancies with clinical indications for chorio-
`nic villus sampling or amniocentesis as per the existing
`obstetric practice of each recruitment unit. Women
`
`were excluded if full karyotyping results were not
`available.
`We recorded the maternal ages at the expected time
`of delivery, gestational ages at blood sampling, and
`indications for invasive testing.
`
`Sample processing
`Peripheral venous blood samples (5–10 mL) were col-
`lected into tubes containing EDTA. A plasma sample
`would be accepted for analysis if it was collected before
`invasive obstetric procedures, was harvested within six
`hours of venepuncture, was at least 2 mL in volume,
`and was not haemolysed. The prospectively collected
`and retrieved archival plasma samples from the UK
`and the Netherlands were sent to Hong Kong in
`batches by overnight courier while kept frozen on dry
`ice. We performed all subsequent analyses prospec-
`tively at The Chinese University of Hong Kong.
`
`Maternal plasma DNA sequencing
`We extracted and sequenced DNA molecules from
`2.0–4.8 mL of maternal plasma using a protocol similar
`to that reported previously but with the introduction of
`multiplexing.24 27 DNA molecules exist in maternal
`plasma as short fragments. The genomic identities of
`millions of DNA molecules per maternal plasma sam-
`ple can be decoded in a sequencing run. The analytical
`goal of our test was to determine the proportion of
`sequenced plasma DNA molecules originating from
`chromosome 21 (percentage chromosome 21). This
`proportion is expected to be elevated in maternal
`plasma during a pregnancy with a trisomy 21 fetus.
`Multiplexing allows more than one plasma sample to
`be mixed and sequenced jointly in each discrete seg-
`ment of a sequencing glass slide and thereby increases
`the number of samples that can be analysed in each
`sequencing run. We studied two levels of multiplexing,
`2-plex and 8-plex, whereby DNA from two or eight
`maternal plasma samples were co-sequenced in each
`slide segment.
`Briefly, a unique synthetic DNA “barcode” of six
`base pairs, referred to as an index, was introduced on
`to one end of each plasma DNA molecule. The index
`served as a signature for a sample, with one index used
`per maternal plasma sample. For example, for 8-plex
`sequencing, eight different indices were needed for
`each of the eight test or control samples that would be
`co-sequenced. We compiled the multiplexed sample
`mixtures by pooling eight or two maternal plasma
`DNA preparations for the 8-plex or 2-plex sequencing
`protocols.
`We performed sequencing on the Genome Analyzer
`II (Illumina) for the 8-plex sample mixtures and on the
`Genome Analyzer IIx (Illumina) for the 2-plex sample
`mixtures. After the sequencing run, the actual DNA
`molecules that belonged to a specific sample could
`then be distinguished from those belonging to other
`samples by sorting the index sequences attached to
`the DNA molecules. We considered a sequencing
`result as valid and reportable only if the analysis of
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`the sample passed a set of quality control measures
`(listed in appendix 1).
`
`Sequencing data analysis
`We processed the sequencing data with the use of a
`bioinformatics algorithm based on our previously
`reported strategy (appendix 2 on bmj.com).24 In
`essence, the chromosomal origin of each sequenced
`“read” (the short fragments of DNA that are decoded)
`was identified by comparing with the reference human
`genome available from the Ensembl website (ftp://ftp.
`ensembl.org/pub/release-48/fasta/homo_sapiens/
`dna/). The percentage chromosome 21 within each
`maternal plasma sample was then calculated. The pro-
`portion of reads from chromosome Y (percentage
`chromosome Y) was similarly calculated and was
`used to estimate the fetal DNA concentrations (that
`is, the proportion of DNA molecules in the maternal
`plasma sample that originated from the fetus) in preg-
`nancies with male fetuses. We derived a z score for
`chromosome 21 in a test sample by subtracting the
`mean percentage chromosome 21 of a reference set
`of euploid pregnancies (controls) from the percentage
`chromosome 21 of the test case and divided by the
`standard deviation of the value for percentage chromo-
`some 21 among the reference sample set according to
`the equation:
`
`Pregnancies with maternal blood sampling (n=824)
`
`Failed recruitment criteria (n=14):
` Twin pregnancy (n=2)
` Without full karyotyping (n=12)
`
`Compromised blood sample (n=46):
` Sample collected after invasive obstetric procedure (n=3)
` Delayed blood processing (n=2)
` With ambiguous information (n=3)
` Haemolysed (n=12)
` Inadequate volume (n=26)
`
`Maternal blood samples (n=764)
`
`Sample failed quality control for sequencing (failed DNA
`extraction, library preparation, or sequencing) (n=11)
`
`Pregnancies with valid results from 8-plex sequencing (n=753)
`
`Euploid fetus (n=597)
`
`Test case (n=501)
`(116 with 2-plex*)
`
`Other fetus:
` Trisomy 18 (n=40)
` (13 with 2-plex*)
` Trisomy 13 (n=20)
` (16 with 2-plex*)
` Turner’s syndrome (n=8)
` (0 with 2-plex*)
` Sex chromosome mosaic (n=2)
` (1 with 2-plex*)
`
`Reference controls (n=96)
`(82 with 2-plex*)
`
`Non-trisomy 21 case (n=571)
`(146 with 2-plex*)
`
`Trisomy 21 fetus (n=86)
`(86 with 2-plex*)
`
`*No of cases with sequencing results from 2-plex protocol
`
`Fig 1 | Recruitment of participants. Numbers in parentheses are the number of cases with
`sequencing results from the 2-plex protocol
`
`RESEARCH
`
`Z score for percentage chromosome 21 in test case =
`((percentage chromosome 21 in test case) − (mean
`percentage chromosome 21 in reference controls))/
`(standard deviation of percentage chromosome 21 in
`reference controls).
`We used a z score of >3 (representing a percentage
`chromosome 21 value greater than that of the 99.9th
`centile of the reference sample set for a one tailed dis-
`tribution) as the cut-off value to determine if the per-
`centage chromosome 21 was increased and hence fetal
`trisomy 21 was present.
`All members of the sequencing and bioinformatics
`teams were blinded to the karyotype information. Dis-
`ease classification based on the sequencing results and
`z scores was generated automatically by the bioinfor-
`matics algorithm. We then determined the diagnostic
`performance of the sequencing test by comparing its
`results with those of full karyotyping of the amniotic
`fluid or chorionic villus sample.
`
`Statistical analysis
`We reported the observed diagnostic sensitivity and
`specificity values of the sequencing protocols based
`on the predefined cut-off point—namely, chromosome
`21 z score of 3. We plotted the receiver operating char-
`acteristic (ROC) curves for the measurements of per-
`centage chromosome 21 and determined the areas
`under the curves (AUC). ROC curve analysis was
`also performed to derive cut-off values for percentage
`chromosome Y to distinguish male and female fetuses.
`We measured the analytical imprecision of the sequen-
`cing protocols by calculating the coefficient of varia-
`tion (SD/mean × 100%)
`of
`the percentage
`chromosome 21 values among the control samples.
`The normality of quantitative variables was analysed
`with the use of the Kolmogorov-Smirnov test. We then
`used Student’s t test or Mann-Whitney test and Pear-
`son’s correlation as appropriate. Two sided P values of
`<0.05 were considered to indicate statistical signifi-
`cance. ROC curve analyses were performed with the
`use of the MedCalc software (version 9.6.4.0). All other
`statistical calculations were performed with the use of
`the SigmaStat software (version 3.11).
`
`RESULTS
`Study participants
`We recruited 576 pregnant women prospectively and
`retrieved archived maternal plasma samples from 248
`women, which amounted to a total of 824 pregnancies
`(fig 1). Fourteen pregnant women (1.7%) did not meet
`the recruitment criteria, and 46 maternal plasma sam-
`ples (5.6%) did not pass the specimen quality require-
`ments. The quality control requirements were not met
`during the sequencing of 11 (1.4%) maternal plasma
`samples (appendix 1). Analytically valid sequencing
`results that met the criteria for reporting were achieved
`for 753 maternal plasma samples with the use of the
`8-plex protocol. The characteristics of these pregnan-
`cies are shown in table 1. The median maternal age was
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`pregnant women underwent combined screening in
`the first
`trimester while the remaining 51 (8.2%)
`women received other forms of prenatal screening,
`including the integrated test and second trimester
`maternal serum screening.
`
`RESEARCH
`
`Table 1 | Characteristics of 753 pregnancies with maternal plasma DNA sequencing. Values
`are numbers (percentages) of subjects unless stated otherwise
`
`Characteristic
`
`Fetal sex:
`
`Male
`
`Female
`
`Sex chromosome mosaic
`
`Maternal age at expected date of
`delivery (years):
`
`<35
`≥35
`Median age
`
`Gestational age at maternal blood
`sampling (weeks+days):
`≤13+6
`>14+0 to 14+6
`
`>15+0
`
`Median gestational age
`
`Means of invasive testing:
`
`Reference
`controls (n=96)*
`
`Non-trisomy 21
`fetuses (n=571)
`
`Trisomy 21
`fetuses (n=86)
`
`All
`(n=753)
`
`96 (100)
`
`0
`
`0
`
`42 (44)
`
`54 (56)
`
`35.7
`
`80 (83)
`
`7 (7)
`
`9 (9)
`
`12+5
`
`251 (44)
`
`318 (56)
`
`2 (0.4)
`
`255 (45)
`
`316 (55)
`
`35.0
`
`410 (72)
`
`80 (14)
`
`81 (14)
`
`13+1
`
`39 (45)
`
`47 (55)
`
`0
`
`22 (26)
`
`64 (74)
`
`38.0
`
`67 (78)
`
`15 (17)
`
`4 (5)
`
`13+0
`
`386 (51)
`
`365 (48)
`
`2 (0.3)
`
`319 (42)
`
`434 (58)
`
`35.4
`
`557 (74)
`
`102 (14)
`
`94 (12)
`
`13+1
`
`621 (82)
`
`Diagnostic performance of maternal plasma DNA
`sequencing
`A mean of 0.3 million (SD 88 000) reads per sample
`from the 8-plex sequencing analysis and 2.3 million
`(SD 474 000) from 2-plex sequencing matched our
`bioinformatics criteria for calculation of percentage
`chromosome 21. As each sequencing glass slide can
`sequence only a finite number of DNA molecules,
`higher levels of multiplexing would lead to fewer
`DNA molecules to be sequenced per sample. Figure
`2 shows the distribution of the chromosome 21 z scores
`among the controls, non-trisomy 21 group, and tris-
`omy 21 group. Table 2 summarises the diagnostic per-
`formance of the sequencing test for detecting fetal
`trisomy 21.
`With a chromosome 21 z score of 3 as the diagnostic
`cut-off point, the 2-plex protocol detected 100% of tris-
`omy 21 fetuses at a 2.1% false positive rate—namely,
`100% sensitivity and 97.9% specificity. This gave a
`positive predictive value of 96.6% and a negative pre-
`dictive value of 100%.
`The 8-plex protocol, however, detected 79.1% of the
`trisomy 21 fetuses at a false positive rate of 1.1%—that
`is, 79.1% sensitivity and 98.9% specificity. The positive
`predictive value was 91.9% and the negative predictive
`
`8-plex sequencing protocol
`
`2-plex sequencing protocol
`
`Controls
`
`Non-trisomy 21
`
`Trisomy 21
`
`10
`
`02468
`
`-2
`
`-4
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`-10
`
`Z score for percentage chromosome 21
`
`Z score for percentage chromosome 21
`
`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
`
`Chorionic villus sampling
`
`Amniocentesis
`
`Risk grouping:
`
`High
`
`Intermediate
`
`Other
`
`Median fetal risk for trisomy 21:
`
`In high risk group
`
`78 (81)
`
`18 (19)
`
`64 (67)
`
`6 (6)
`
`26 (27)
`
`1 in 50
`
`In intermediate risk group
`1 in 486
`*Euploid male fetuses. NA=not applicable.
`
`462 (81)
`
`109 (19)
`
`437 (77)
`
`33 (6)
`
`101 (18)
`
`1 in 51
`
`1 in 513
`
`81 (94)
`
`5 (6)
`
`81 (94)
`
`0
`
`5 (6)
`
`1 in 2
`
`NA
`
`132 (18)
`
`582 (77)
`
`39 (5)
`
`132 (18)
`
`1 in 43
`
`1 in 502
`
`35.4 years, and the median gestational age at the time
`of maternal blood sampling was 13 weeks and 1 day.
`Full karyotyping showed that there were 86 preg-
`nancies with trisomy 21 fetuses (40 cases from the
`archival sample set) and 597 pregnancies with euploid
`fetuses (fig 1). The remainder comprised 40 fetuses
`with trisomy 18, 20 with trisomy 13, eight with Turn-
`er’s syndrome, and two sex chromosome mosaics, all
`of which were included as non-trisomy 21 cases in this
`study.
`Among the sequenced samples, we randomly chose
`96 pregnancies with euploid male fetuses as the refer-
`ence control set for z score calculation. All 86 trisomy
`21 cases, 82 reference controls, and 146 non-trisomy
`21 cases were sequenced with the 2-plex protocol
`(fig 1).
`Review of the clinical profile of the cases revealed
`there were three broad groups of clinical indications
`for referral for full karyotyping: (a) pregnancies with
`a risk higher than 1 in 300 for fetal trisomy 21 as esti-
`mated by conventional prenatal screening; (b) preg-
`nancies with an intermediate risk (risk between 1 in
`300 and 1 in 1000) for fetal trisomy 21; and (c) pregnan-
`cies with other risk indications, including a previous
`trisomy 21 pregnancy, ultrasound abnormalities, or
`risks for monogenic diseases. The numbers of pregnan-
`cies in the high, intermediate, and other risk groups
`were 582, 39, and 132, respectively (table 1). Among
`the high and intermediate risk groups, 570 (91.8%)
`
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`Table 2 | Diagnostic performance of maternal plasma DNA sequencing for detecting fetal trisomy 21 and fetal sex
`
`8-plex sequencing protocol
`
`2-plex sequencing protocol
`
`True detection rate
`
`False positive rate
`
`True detection rate
`
`False positive rate
`
`Trisomy 21 detection*
`
`Among 86 trisomy 21 cases
`
`Among 571 non-trisomy 21 cases
`
`Among 86 trisomy 21 cases
`
`Among 146 non-trisomy 21 cases
`
`Fetal sex (male) detection††
`
`Among 386 male fetuses
`
`Among 365 female fetuses
`
`Among 196 male fetuses
`
`Among 117 female fetuses
`
`79.1% (68/86)
`
`1.1% (6/571)
`
`100% (86/86)
`
`2.1% (3/146)
`
`99.5% (384/386)
`(95% CI 98.1% to 99.9)
`
`0.8% (3/365)
`(95% CI 0.2% to 2.4%)
`
`99.5% (195/196)
`(95% CI 97.2% to 99.9%)
`
`0.8% (1/117)
`(95% CI 0.1% to 4.7%)
`
`*Z score for percentage chromosome 21 >3.
`†Cut-off values for percentage chromosome Y identified by ROC analysis.
`
`protocol, the post-test probabilities for a positive result
`and a negative result becomes 1 in 16 and 1 in 5082
`respectively.
`These data suggest that the sequencing test result,
`whether positive or negative, can substantially alter
`the probabilities for having a trisomy 21 fetus. The 2-
`
`8-plex sequencing protocol
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`100
`
`2-plex sequencing protocol
`
`80
`
`60
`
`40
`
`20
`
`0
`0
`
`20
`
`40
`
`60
`
`80
`
`100
`
`100 - (specificity (%))
`
`Sensitivity (%)
`
`Sensitivity (%)
`
`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
`
`value was 96.9%. Eleven of the 18 false negative cases
`were from the archived samples. The distribution of
`the false negative and true positive cases among the
`prospectively collected and archived samples was not
`statistically significant (χ2 test, P=0.258). The median
`gestational ages for the true positive and false negative
`trisomy 21 cases from the 8-plex protocol were
`(interquartile range 12.3–13.5) and
`13.0 weeks
`12.9 weeks (12.3–13.5), respectively. The median risk
`level for trisomy 21 estimated by conventional screen-
`ing for the true positive and false negative cases were 1
`in 4 (interquartile range 1 in 5 to 1 in 2) and 1 in 5 (1 in 7
`to 1 in 2), respectively. The true positive and false nega-
`tive cases were not significantly different in terms of
`gestational ages (Mann-Whitney, P=0.538) or risk
`levels (Mann-Whitney, P=0.466). There were six false
`positive results in the 8-plex results and three in the
`2-plex results. Because of the small number of false
`positive cases, no systematic features were identified.
`Profiles of these cases are shown in appendix 3 on
`bmj.com.
`ROC curve analysis of the percentage chromosome
`21 measurements for all test cases was done to estimate
`the diagnostic efficacy of the 2-plex and 8-plex sequen-
`cing protocols (fig 3). Area under the curve values for
`the 2-plex and 8-plex sequencing protocols were 1.00
`(95% confidence interval 0.98 to 1.00) and 0.98 (0.97 to
`0.99) respectively.
`
`Utility of maternal plasma DNA sequencing for the prenatal
`assessment of trisomy 21
`In this study we focused on pregnancies clinically indi-
`cated for invasive prenatal diagnostic testing because
`we required the availability of full karyotyping for
`comparison of
`the diagnostic performance of
`the
`sequencing test. However, this study design essentially
`limits the applicability of our data to pregnancies at
`increased risk for trisomy 21. To investigate if the
`sequencing test might be useful as a screening test for
`all pregnancies in general, we determined the post-test
`probabilities for women of different ages and hence
`with different prevalences of trisomy 21.28 The data
`are shown in table 3. For a 20 year old woman at
`12 weeks of gestation, her pretest probability for tris-
`omy 21 by maternal age alone was 1 in 1068. If mater-
`nal plasma DNA sequencing with the 2-plex protocol
`was done, the post-test probability for a positive test
`result was 1 in 23 while the post-test probability for a
`negative test result was 1 in infinity. With the 8-plex
`
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`plex sequencing particularly highlights the key value
`of the sequencing test result for ruling out trisomy 21.
`
`Factors affecting diagnostic performance
`The diagnostic performance of the 2-plex protocol was
`superior to that of the 8-plex sequencing, with signifi-
`cantly higher z score values for the trisomy 21 cases
`(Mann-Whitney, P<0.001). The high z scores were
`due to a small standard deviation and, hence, better
`precision for estimating percentage chromosome 21.
`The coefficients of variation of percentage chromo-
`some 21 values for the controls on each flow cell
`were calculated as a measure of precision (see appen-
`dix 4 on bmj.com). The mean coefficient of variation
`for measuring percentage chromosome 21 with the
`8-plex protocol was 1.59% (SD 0.36%) and was higher
`than that for the 2-plex protocol, which was 0.66% (SD
`0.25%) (t test, P<0.001).
`The extent of increase in percentage chromosome
`21 in maternal plasma for the trisomy 21 cases is gov-
`erned by the fetal DNA concentration. We calculated
`the fetal DNA concentrations based on the percentage
`chromosome Y values among the 39 male fetuses with
`trisomy 21 and plotted them against the chromosome
`21 z scores (shown in appendix 5 on bmj.com). The two
`variables are significantly correlated in both the 8-plex
`sequencing data (Pearson correlation coefficient 0.703,
`P<0.0001) and 2-plex data (Pearson correlation coeffi-
`cient 0.462, P=0.003). The five pregnancies with male
`fetuses with trisomy 21 that we failed to detect using the
`8-plex protocol (that is, with chromosome 21 z scores
`<3) had fetal DNA concentrations less than 10%.
`Theoretically, at 10% fetal DNA concentration, per-
`centage chromosome 21 is just 1.05 times higher in a
`plasma sample obtained from a trisomy 21 pregnancy
`than a euploid pregnancy.18 To detect this increase in
`
`percentage chromosome 21, the coefficient of varia-
`tion of the test would need to be ≤0.83% (see appendix
`6 on bmj.com). The mean coefficient of variation of the
`8-plex protocol was greater than this value, whereas
`that of the 2-plex protocol was <0.83%. Hence, the
`2-plex protocol performed better at detecting trisomy
`21 among samples containing low fetal DNA concen-
`trations.
`
`Fetal sex determination by maternal plasma DNA
`sequencing
`The presence of chromosome Y sequences among
`sequenced DNA molecules from maternal plasma
`should theoretically signify that
`the pregnancy
`involved a male fetus. However, we previously
`reported that
`the bioinformatics algorithm would
`incorrectly assign a small fraction of sequenced DNA
`reads to chromosome Y in pregnancies with female
`fetuses.24 The percentage chromosome Y values of all
`studied pregnancies are shown in appendix 7 on
`bmj.com. Optimal cut-off values for percentage chro-
`mosome Y to distinguish male and female fetuses were
`identified by ROC curve analysis to be 0.0114% and
`0.0095% for the 8-plex and 2-plex protocols, respec-
`tively. Table 2 shows the estimated diagnostic perfor-
`mance
`for
`fetal
`sex determination. We
`then
`determined the fetal DNA concentrations of 314 preg-
`nancies with euploid male fetuses based on the percen-
`tage chromosome Y values obtained from the 8-plex
`protocol. The median fetal DNA concentration was
`15.2% (interquartile range 10.6%–19.1%), and the dis-
`tribution is shown in fig 4. Among the pregnancies with
`euploid male fetuses, the median fetal DNA concentra-
`tion for the archival and prospectively collected sam-
`ples were 14.7% and 15.4%, respectively (Mann-
`Whitney, P=0.334).
`
`Table 3 | Probabilities for a trisomy 21 fetus in women by age alone and according to result of maternal plasma DNA
`sequencing test
`
`Maternal age
`(years)
`
`Pretest probability*
`
`8-plex sequencing
`Positive test result†
`
`Negative test result
`
`2-plex sequencing
`Positive test result†
`
`Negative test result
`
`Post-test probability
`
`20
`
`25
`
`30
`
`31
`
`32
`
`33
`
`1 in 1068
`
`1 in 946
`
`1 in 626
`
`1 in 543
`
`1 in 461
`
`1 in 383
`
`1 in 16
`
`1 in 14
`
`1 in 10
`
`1 in 9
`
`1 in 7
`
`1 in 6
`
`1 in 5
`
`1 in 5082
`
`1 in 4501
`
`1 in 2977
`
`1 in 2582
`
`1 in 2191
`
`1 in 1820
`
`1 in 1482
`
`1 in 23
`
`1 in 21
`
`1 in 14
`
`1 in 12
`
`1 in 11
`
`1 in 9
`
`1 in 8
`
`1 in infinity
`
`1 in infinity
`
`1 in infinity
`
`1 in infinity
`
`1 in infinity
`
`1 in infinity
`
`1 in infinity
`
`34
`
`35
`
`36
`
`37
`
`38
`
`39
`
`40
`
`41
`
`42
`
`1 in 312
`
`1 in 249
`
`1 in 196
`
`1 in 152
`
`1 in 117
`
`1 in 89
`
`1 in 68
`
`1 in 51
`
`1 in 38
`
`1 in 4
`
`1 in 4
`
`1 in 3
`
`1 in 3
`
`1 in 2
`
`1 in 2
`
`1 in 2
`
`1 in 2
`
`1 in 1182
`
`1 in 930
`
`1 in 720
`
`1 in 553
`
`1 in 420
`
`1 in 320
`
`1 in 239
`
`1 in 177
`
`1 in 6
`
`1 in 5
`
`1 in 4
`
`1 in 3
`
`1 in 3
`
`1 in 2
`
`1 in 2
`
`1 in 2
`
`1 in infinity
`
`1 in infinity
`
`1 in infinity
`
`1 in infinity
`
`1 in infinity
`
`1 in infinity
`
`1 in infinity
`
`1 in infinity
`
`*Pretest probabilities are based on prevalence of fetal trisomy 21 at 12th week of gestation.28
`†A positive test result is a sample with a z score for percentage chromosome 21 >3.
`
`page 6 of 9
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`RESEARCH
`
`prospectively collected and archived maternal plasma
`samples were processed using the same protocols and
`analysed prospectively. There were also no significant
`differences in the fetal DNA concentrations and test
`performances between the two sample groups. In this
`study 5.6% of the collected maternal plasma samples
`were of compromised quality. Reportable sequencing
`results were not achieved for 11 samples. All these sam-
`ples were identified by the predefined quality control
`steps. In routine practice, laboratory reports would not
`be issued for such samples, and another blood speci-
`men would be requested.
`We found other aneuploidies besides trisomy 21
`among the recruited pregnant women. For this study,
`we focused on the diagnostic performance of the
`sequencing approach for fetal trisomy 21 because our
`previous data showed that the measurements of the
`proportion of DNA molecules from chromosomes 18
`and 13 were much less precise.24 25 More research is
`required to develop protocols to improve the precision
`for measuring amount of DNA molecules from chro-
`mosomes 18 and 13.29
`Our data reveal that the main value of the maternal
`plasma DNA sequencing test is to rule out fetal trisomy
`21. Hence, with the current diagnostic performance, it
`is more suitable as a screening test to stratify pregnan-
`cies whose risk for trisomy 21 warrants the considera-
`tion of amniocentesis or chorionic villus sampling. In
`this study, we performed the sequencing test after a
`pregnancy was deemed to be at increased risk for tris-
`omy 21 according to the current prenatal screening
`programmes. The false positive rates of the current
`screening programmes are about 5%, and all of these
`pregnanct women are offered the option of invasive
`testing. However, if we took into consideration the
`results of the sequencing test, trisomy 21 could be
`ruled out in 98% of those pregnancies. This would
`leave just 0.1% (that is, 5%×(100%−98%)×100%) of all
`pregnant women needing referrals for amniocentesis
`or chorionic villus sampling. Most of our studied preg-
`nancies were in the first trimester, which suggests that it
`is possible to implement the test even in early preg-
`nancy as a second tier screening test after the first tri-
`mester combined test, which is already in use in many
`parts of the world.
`On the other hand, our post hoc analysis shows that
`the sequencing test result may alter clinical decisions
`even in women at