DOI 10.1515/labmed-2012-0029 
`
` J Lab Med 2012; 36(5): 269–275
`
` Molekulargenetische und zytogenetische Diagnostik/
`Molecular-Genetic and Cytogenetic Diagnostics
`
`Redaktion: H.-G. Klein
`
` Amy Swanson, Christin Coffeen and Amy J. Sehnert *
` Non-invasive prenatal testing for fetal aneuploidy
`by massively parallel DNA sequencing of maternal
`plasma: the future has arrived today
`
` Nicht-invasiver Pr ä natal-Test auf fetale Aneuploidie mittels massiv-paralleler DNA-
`Sequenzanalyse im m ü tterlichen Plasma: in der Zukunft angekommen
`
` Abstract : After decades of research, non-invasive prena-
`tal 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 dem-
`onstrated 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 technol-
`ogy and key considerations for clinical implementation
`are discussed.
`
` Keywords: cell-free DNA (cfDNA); fetal aneuploidy; mas-
`sively parallel sequencing (MPS); maternal plasma; non-
`invasive prenatal testing (NIPT) .
`
`l ö st einen lange herbeigesehnten Paradigmenwechsel in
`der pr ä natalen Diagnostik aus. Es wurde bereits eine Viel-
`zahl von Methoden, welche die Sequenzanalyse von m ü t-
`terlicher zell-freier DNA (cfDNA) verwenden, untersucht.
`Dabei zeigte sich, dass dieser Ansatz f ü r den Nachweis
`der h ä ufigsten fetalen Aneuploidie, der Trisomie 21 (T21)
`genutzt werden kann. Der Fokus dieses Artikels liegt auf
`der massiv-parallelen Sequenzanalyse (MPS) mit optimier-
`tem „Sequence Tag Mapping “ und Chromosomenquantifi-
`zierung, wodurch T21 und zahlreiche andere Aneuploidie
`im Genom exakt nachgewiesen werden k ö nnen. Der Vorteil
`dieser Methode liegt in ihrer hohen Pr ä zision und der Redu-
`zierung der Variation innerhalb eines sowie zwischen meh-
`reren Sequenzierungsl ä ufen. Durch die Verwendung von
`MPS kann der Aneuploidie-Status einer Probe zuverl ä ssig
`allein aus der genetischen Information ohne die Fakturie-
`rung in anderen m ü tterlichen Pr ä test-Risiko oder anderen
`klinischen Variablen ermittelt werden. Die Eignung dieser
`Methode konnte prospektiv in einer streng verblindeten,
`multizentrischen Studie in den USA demonstriert werden.
`Die Ergebnisse deuten darauf hin, dass MPS in existierende
`pr ä natale Screening-Algorithmen integriert werden kann
`und somit unn ö tige invasive Eingriffe reduziert werden
`k ö nnen. Die Technologie und ihre klinische Implementie-
`rung werden diskutiert.
`
` Schl ü sselw ö rter:
` massiv-parallele
` fetale Aneuploidie;
`Sequenzanalyse (MPS); m ü tterliches Plasma; Nicht-inva-
`siver Pr ä natal-Test (NIPT); zellfreie DNA (cfDNA).
`
` Zusammenfassung : Nach jahrzehntelanger Forschung hat
`der Nicht-invasive Pr ä natal-Test (NIPT) des fetalen Chromo-
`somenstatus im m ü tterlichen Plasma seinen Weg aus den
`Forschungslaboren in die klinische Praxis gefunden und
`
` *Correspondence: Amy J. Sehnert, MD, Clinical Affairs
`Department, Verinata Health, Inc., 800 Saginaw Drive,
`Redwood City, CA 94063, USA, Tel.: + 1-650-503-5213,
`Fax: + 1-650-362-2314, E-Mail: asehnert@verinata.com
`
`00001
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`270
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` Swanson et al.: Non-invasive prenatal testing by massively parallel sequencing
`
` Amy Swanson: Department of Clinical Affairs , Verinata Health, Inc.,
`Redwood City, CA , USA
` Christin Coffeen: Department of Clinical Affairs , Verinata Health,
`Inc., Redwood City, CA , USA
`
` Introduction
` Fetal chromosome abnormalities are a major contribu-
`tor to miscarriages, congenital anomalies and perinatal
`deaths [1, 2] . Since the 1970s, when amniocentesis was
`first introduced, followed by the introduction of chori-
`onic villus sampling (CVS) in the 1980s, pregnant women
`have had options to obtain information about fetal chro-
`mosome status [3] . Cytogenetic karyotyping of fetal cells
`or chorionic villi obtained from these procedures leads
`to diagnosis in the vast majority of cases with very high
`sensitivity and specificity ( ∼ 99 % ) when adequate tissue
`is obtained [4, 5] . However, these procedures also pose
`risks to the fetus and pregnant woman [6, 7] . To mitigate
`these risks, a series of prenatal screening algorithms
`have been developed to stratify women for their likeli-
`hood of the most common fetal trisomies, trisomy 21
`(T21, Down syndrome), trisomy 18 (T18, Edwards syn-
`drome) and, to a lesser extent, trisomy 13 (T13, Patau syn-
`drome). The screens involve measurement of multiple
`biochemical analytes in the maternal serum at different
`time points combined with ultrasonographic measure-
`ment of the fetal nuchal translucency (NT) and incorpo-
`ration of other maternal factors, such as age, to generate
`a risk score. Based on their development and refinement
`over the years and depending on when the screening is
`administered (first or second trimester only, sequential,
`or fully integrated) and how the screening is adminis-
`tered (serum-only or serum combined with NT), a menu
`of options has evolved with variable detection rates
`(65 % –90 % ) and high screen positive rates (5 % ) [3] . For
`patients, following this multi-step process, the resultant
`non-definitive “ risk score ” can be confusing and anxiety
`provoking, particularly in the absence of comprehen-
`sive counseling [8] . Ultimately, the results are weighed
`against the risks for miscarriage from an invasive pro-
`cedure in a woman ’ s decision-making. For decades,
`beginning with the pursuit of fetal cells in the maternal
`circulation, a better non-invasive means to obtain more
`definitive information on fetal chromosomal status had
`been sought [9] .
`
` Massively parallel sequencing of
`maternal plasma DNA
` Fan et al. were the first to demonstrate counting chromo-
`somes by mapping sequence tags generated by massively
`parallel sequencing (MPS) as a potential quantification
`method for detecting fetal aneuploidy from total cell-free
`DNA (cfDNA) in maternal plasma [10] . Since this seminal
`paper, this method has been optimized and proven to
`be robust for detecting multiple chromosome abnor-
`malities in hundreds of samples from two independent
`studies [11, 12] . Other studies have also demonstrated
`performance of the method for detection of T21 [13 – 15] .
`Maternal plasma isolated from a single 10 mL blood
`tube provides sufficient cfDNA for random sequencing
`analysis, generating tens of millions of sequence tags
`across the entire genome that can be uniquely aligned
`and counted [11] . The plasma is a mixture of maternal
`and fetal cfDNA, and the percent contributed by the
`fetus is referred to as the fetal fraction. In the presence
`of fetal aneuploidy, sequencing produces an increase or
`decrease in the relative number of tags on the affected
`chromosome compared to the euploid chromosomes.
`Calculating and comparing the relative number of tags
`between affected and unaffected chromosomes as
`described below leads to accurate classification of ane-
`uploidy status. Analysis methods have approximately 10
`million tags per sample to maintain high precision down
`to low levels of fetal fraction (approximately 3 % – 4 % )
` [16] . Importantly, improvements in MPS efficiencies have
`increased sequencing depth such that multiple samples
`can now be analyzed per lane (e.g., 6 – 12 samples per
`lane, referred to as a 6-plex or 12-plex), while preserv-
`ing the necessary counting statistics to accurately detect
`fetal aneuploidy [12] . These efficiencies are expected to
`continue, along with decreasing sequencing costs and
`shorter run times, keeping MPS on the forefront as a
`robust and cost-effective platform for non-invasive pre-
`natal testing (NIPT) with genome-wide capability [17] .
` No enrichment strategies (with their concomitant
`increase in error) are required to produce adequate sta-
`tistics. When compared to current chromosome-selective
`sequencing methods [18] , MPS offers several advantages,
`including removal of inherent bias created by selec-
`tive primers, avoidance of the need for target enrich-
`ment strategies to produce adequate allelic counts, no
`requirement to incorporate the fetal DNA fraction in the
`final diagnostic algorithms and higher precision allow-
`ing definitive aneuploidy classification vs. “ risk score ”
`assignment [19] .
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` To begin to test samples for aneuploidy status by this
`method, the following steps are taken. The averages and
`standard deviations of chromosome ratios for the unaf-
`fected samples in the training set are determined, and for
`each test sample, a normalized chromosome value (NCV)
`is calculated using the equation:
`
`j
`
`σ−
`
`ˆ
`
`ˆ
`μ
`j
`
`x
`
`ij
`
`NCV
`ij
`
`=
`
` where μˆ j and σˆ j are the estimated mean and standard
`deviation of the ratios in the training set, respectively,
`for the j- th chromosome ratio, and x ij is the observed j -th
`chromosome ratio for the test sample, i . When chromo-
`some ratios are normally distributed, the NCV is equiva-
`lent to a statistical Z-score for the ratios. Moving from NCV
`score to classification of the autosomes ’ aneuploidy state,
`we require an NCV > 4.0 to classify the chromosome as
`affected (i.e., aneuploidy detected for that chromosome)
`and an NCV < 2.5 to classify a chromosome as unaffected
`(aneuploidy not detected). Samples with autosomes that
`have an NCV between 2.5 and 4.0 are unclassifiable. We
`intentionally maintain the “ unclassifiable ” zone to ensure
`a safe and effective test result. In other words, if there is an
`equivocal value from MPS, we want to be able to identify
`it as such. At the same time, this also allows greater confi-
`dence in sample results that lie in the “ detected ” (NCV > 4)
`and “ not detected ” (NCV < 2.5) zones.
` The classification strategy for sex chromosomes
`is somewhat more complex and described in detail by
`Bianchi et al. [12] . In addition to the high sensitivity and
`specificity for detection of T21, T18 and T13, the NCV
`method also demonstrates high performance to detect
`monosomy X (45,X, Turner syndrome). This condition,
`which is not included in current conventional prenatal
`screens, results in a high miscarriage rate and can present
`at birth with life-threatening cardiac defects and other
`significant medical issues. The demonstration of MPS to
`detect monosomy X offers a potential new avenue for early
`prenatal diagnosis of this condition, which occurs even
`more frequently than T13. And when used in cases where
`fetal cystic hygroma is seen by ultrasound, monosomy X is
`more commonly detected than T18.
`
` Optimized chromosome
`quantification
` Intra-run and inter-run sequencing variation in the chro-
`mosomal distribution of sequence reads can obscure the
`effects of fetal aneuploidy on the distribution of mapped
`sequence sites. Several publications in 2011 noted this
`difficulty in determining aneuploidy in chromosomes
`18 and 13, for example [13, 14] . To correct for such vari-
`ation, we have utilized a chromosome ratio, where the
`count of mapped sites for a given chromosome of interest
`(e.g., chromosome 21) is normalized to cumulative counts
`observed on a predetermined set of custom chromosomes.
`We refer to this predetermined set of chromosomes as
`the “ reference chromosomes ” , and they are used in the
`denominator for the ratio calculations. This approach mit-
`igates the need to perform additional corrections on the
`data [e.g., correction for guanine-cytosine (GC) content
`used by others] (Figure 1 ) [20, 21] . The optimal set of ref-
`erence chromosomes for each chromosome of interest
`is determined from results of data on a training set that
`includes only euploid samples (diploid karyotype). Any
`combination of autosomes other than chromosomes 21, 18
`and 13 are considered as potential denominators in a ratio
`of counts with the chromosomes of interest. To achieve
`the highest precision, denominator chromosomes were
`determined that minimize the variation of the chromo-
`some ratios within and between sequencing runs [11, 12] .
`Using this approach, any chromosome can be analyzed in
`the numerator as the “ chromosome of interest ” , allowing
`whole genome interrogation.
`
`Cell-free DNA
`sequenced and aligned
`to Genome
`
`NCV method
`
`# Counts Chr 21
`
`# Counts on custom
` reference Chr(s)
`
`Z-score method
`
`# Counts Chr 21
`
`# Counts on all
`chromosomes
`
`GC correction
`
`NCV result
`
`Z-score result
`
` Figure 1   Differences between sequencing analysis approaches
`for the normalized chromosome value (NCV) method vs. Z-score
`method with guanine-cytosine (GC) correction.
` The NCV method uses reference chromosomes to reduce variability
`and maximize precision and dynamic range between affected and
`unaffected samples.
`
` Importance of study design
` Early studies in the field led to some urgency to perform
`large-scale properly conducted clinical evaluation studies
`of NIPT [22] . Since then, the results of several independ-
`ent large studies have now been reported [12, 15, 20, 23] .
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`When comparing results from these studies, it is not only
`important to understand their underlying technical dif-
`ferences, but to also recognize the value of study design
`on the strength of the results before transitioning to
`clinical use. The M at E rnal BLood IS S ource to A ccurately
`Diagnose Fetal Aneuploidy (MELISSA) study, reported by
`Bianchi et al., was a prospective, blinded study designed
`to emulate real-world testing in which fetal chromosome
`status is not known a priori [12] . In this study, all samples
`with any abnormal karyotype were included for analysis,
`sequenced and assigned a ploidy status (affected, unaf-
`fected or unclassified) for each of six independent catego-
`ries: chromosome (Chr) 21, Chr 18, Chr 13, male, female
`and monosomy X. The ratio and frequency of abnormal
`to normal samples was unknown, and no maternal clini-
`cal factors were used in the classification of samples.
`The importance of this design is that for each of the six
`independent categories analyzed, all samples that did
`not have the test condition (e.g., aneuploidy 18 for Chr
`18), served as controls in the analysis. For example, after
`unblinding, it was shown that a very diverse set of karyo-
`types was represented in 35 % of the “ non-aneuploidy 18 ”
`population. In other words, non-aneuploidy 18s included
`samples with other major aneuploidies (e.g., T21) and
`other chromosome abnormalities as would be expected
`to be encountered in real-world testing (Figure 2 ). This is
`an important distinction that has not been demonstrated
`in any other studies to date. Under these conditions, the
`test achieved 100 % specificity, no false-positives, with
`a narrow 95 % confidence interval range (99.1 % – 100 % )
`and very high sensitivity (Chr 21 100 % , 95 % CI 95.9 – 100;
`Chr 18 97.2 % , 95 % CI 85.5 – 99.9 and Chr 13 78.6 % , 95 % CI
`49.2 – 95.3). For monosomy X, 15 of 16 cases were detected
`(sensitivity 93.8 % , 95 % CI 69.8 – 99.8). All results showed
`
`A
`
`Others
`
`B
`
`MELISSA
`
`Euploid
`
`superior sensitivity and specificity compared to serum
`analytes and ultrasound.
` Another distinction of this study was that all chromo-
`somes were examined for each sample. This analysis led
`to the correct identification of two cases of other autoso-
`mal aneuploidies (T20 and T16) and several cases of sex
`chromosome aneuploidies (XXX, XXY and XYY). Thus,
`MPS offers a whole-genome approach without having
`to design specific primers – as would be required with
`chromosome-selective sequencing analysis. From a clini-
`cal standpoint, this is very important and offers future
`development of the technology to meet a broader number
`of conditions.
` Lastly, the MELISSA study also included samples from
`pregnant women who conceived by in vitro fertilization,
`some of whom had pregnancies affected by aneuploidy.
`These results suggest that NIPT by MPS is also accurate in
`this group of women who may particularly wish to avoid
`an invasive procedure if possible.
`
` Clinical use
` The overall goal of NIPT is to minimize anxiety surround-
`ing multi-step screening and reduce false-positive results,
`thereby reducing exposure to invasive procedural risks.
`This goal is finally being realized through the introduction
`of laboratory developed prenatal MPS testing. In response
`to these offerings, the International Society of Prena-
`tal Diagnosis (ISPD) and the National Society of Genetic
`Counselors (NSGC, USA) have issued rapid response and
`position statements on the topic [24, 25] . ISPD accepts that
`with suitable genetic counseling MPS can be helpful for
`
`Euploid
`
`Trisomy 21
`
`Trisomy 13
`
`Monosomy X
`
`Sex aneuploidy
`
`Translocations
`
`Other rare autosomal
`aneuploidies
`Others-mosaicism and
`complex variants
`
` Figure 2   Differences in control populations used for specificity analysis.
` Panel A shows a fully euploid population used in all prior studies. Panel B shows diverse non-T18 karyotypes included in Chr 18 analysis by
`Bianchi et al. [12] .
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`
`women who may have been determined to be high-risk
`by one of the previously recommended screening strate-
`gies. Based on the peer-reviewed evidence, NSGC also
`recognizes and supports NIPT as an option for aneuploidy
`assessment of chromosomes 21, 18 and 13 in pregnancy
`for high-risk women. They suggest that any abnormal
`results should be confirmed through a conventional pre-
`natal diagnostic procedure. NSGC also recommends both
`pre- and post-test genetic counseling to provide up-to-
`date information and ensure patient understanding of the
`limitations of the test and implications of the results (see
`below). Neither ISPD nor NSGC currently supports NIPT as
`a routine, first-tier aneuploidy screening test in low-risk
`populations until further evidence is provided.
` While these initial steps of implementation and intro-
`duction of NIPT into clinical practice have quickly taken
`place, it is clear that ongoing education is needed for
`healthcare providers and patients alike to fully under-
`stand all the ramifications of testing. In individual situ-
`ations, there may be certain scenarios where a pregnant
`woman may request NIPT as an alternative to conven-
`tional screening, an invasive prenatal procedure or both.
`It is particularly important in these cases that comprehen-
`sive counseling be provided.
`
` Genetic counseling considerations
` By offering NIPT as a secondary screen to those women
`with a positive conventional screen (or other a priori risk),
`the number of unnecessary amniocentesis and CVS proce-
`dures are expected to decrease. Conversely, the need for
`genetic counseling will increase, as informed consent is a
`critical component of NIPT. Resource limitations to meet
`this demand need to be considered within practices to
`ensure that a qualified healthcare provider (if not a certi-
`fied genetic counselor) is available to provide non-direc-
`tive pre-test counseling for all women considering the test.
`As a positive NIPT result is more similar to a positive result
`from amniocentesis or CVS, women should be given the
`opportunity prior to this testing to decide whether they
`desire this degree of information. Pre-test genetic coun-
`seling for NIPT should also include discussion of the rec-
`ommendation for confirmation of abnormal test results
`via CVS or amniocentesis (depending upon gestational
`age) so that appropriate consideration can be given to the
`expected timing of results for post-test planning. Per the
`NSGC statement, because NIPT does not currently screen
`for all chromosomal or genetic conditions, it does not
`replace standard risk assessment and prenatal diagnosis.
`
`Patients with other factors (e.g., abnormal ultrasound
`findings) suggestive of a chromosome abnormality should
`receive genetic counseling and have the option of con-
`ventional diagnostic testing, regardless of NIPT results.
`Women should also be made aware that for some patients
`an NIPT result may not be informative.
`
` Biological considerations
` NIPT is perhaps more similar to CVS than amniocentesis,
`in that detection of aneuploidy is typically representative
`of the chromosomal constitution of the fetus, but in some
`instances may be representative of confined placental
`aneuploidy or confined placental mosaicism (CPM). CPM
`occurs in approximately 1 % – 2 % of cases of CVS results
`today, and some women undergo an amniocentesis at
`a later gestational age after CVS to make the distinction
`between apparently isolated placental aneuploidy vs.
`fetal aneuploidy. As NIPT is implemented more widely,
`cases of CPM are expected to cause some number of posi-
`tive NIPT results that may not be subsequently confirmed
`by an invasive procedure, particularly amniocentesis.
`Further investigation of the pregnancy course and out-
`comes as well as placental pathology will be important
`in these cases. Similarly, NIPT also has the potential to
`identify maternal full or mosaic aneuploidy given that
`total cfDNA is a mixture of maternal and fetal DNA. These
`and other conditions, such as low-level fetal mosaicism
`and undetected multiples or demised co-twins, need to be
`considered in the overall clinical context.
`
` Clinical test
` Verinata Health, Inc. (Redwood City, CA, USA), began
`offering the verifi ® prenatal test in early 2012 in the US for
`detection of aneuploidies in chromosomes 21, 18 and 13
`from a single maternal blood draw as early as 10-weeks ’
`gestation. The test is currently indicated for women
`with singleton pregnancies and high-risk indications for
`fetal aneuploidy. The test is available through Verinata ’ s
`College of American Pathologists (CAP) accredited clinical
`laboratory. Results are reported as “ Aneuploidy Detected ” ,
` “ No Aneuploidy Detected ” , or “ Unclassifiable ” for each of
`the three chromosomes evaluated. An option for mono-
`somy X detection in patients with findings of fetal cystic
`hygroma or increased nuchal translucency has recently
`been added. Physician signature and patient informed
`consent are required for testing.
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`
` Future considerations
` All clinical studies to date have focused on pregnant
`women with high-risk indications. Although there are
`few reasons to believe that MPS performance will differ
`significantly in low-risk populations, several studies are
`currently underway to test this hypothesis. As clinical
`experience accumulates, and if results are successful from
`comparative studies to conventional screening, it is possi-
`ble that NIPT will replace current screening protocols and
`possibly serve as a primary tool.
` Another area for future investigation includes studies
`on pregnancies with multiple gestations. The majority of
`studies to date have been performed on singleton preg-
`nancies, although Sehnert et al. previously reported on the
`correct classification of five twin gestations (two affected,
`three unaffected) [11] and Canick et al. recently reported
`data from 25 twin gestations (eight with at least one affected
`fetus) [26] . Although these results are encouraging, larger
`sample sizes are needed to accurately determine the per-
`formance of maternal cfDNA sequencing for multiple
`gestations.
` Finally, a few examples of detecting partial chro-
`mosome aneuploidy have been shown feasible and
`
`demonstrate the potential of MPS to non-invasively deter-
`mine molecular karyotype [11, 12, 27, 28] . Future research is
`aimed at further investigating the ability of this technology
`to detect sub-chromosomal deletions and duplications.
`
` Conclusions
` Developments in the field of NIPT are exciting and rapidly
`evolving and promise positive changes for prenatal care.
`Cumulative evidence suggests that NIPT using MPS can
`be safely introduced into existing prenatal screening
`algorithms to reduce unnecessary invasive procedures.
`
` Conflict of interest statement
` Authors ’ conflict of interest disclosure: The authors stated that we
`are all employees of Verinata Health regarding the publication of this
`article.
` Research funding: None declared.
` Employment or leadership: The authors are employees of Verinata
`Health, Inc., which is a for-profit company providing pre-natal diag-
`nostic service using MPS in NIPT.
` Honorarium: None declared.
`
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