Determination of fetal chromosome aberrations from fetal DNA in maternal blood: has the challenge finally been met?

2011·0.26 MB·English

by  Hahn, Sinuhe

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Determination of fetal chromosome e
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aberrations from fetal DNA in Dn
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maternal blood: has the challenge e
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finally been met? on
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Sinuhe Hahn1,*, Olav Lapaire2, Sevgi Tercanli2, Varaprasad Kolla1 on
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and Irene Hösli2 t
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The analysis of cell-free fetal nucleic acids in maternal blood for prenatal rh
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diagnosis has been transformed by several recent profound technology b
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developments. The most noteworthy of these are ‘digital PCR’ and ‘next-
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generation sequencing’ (NGS), which might finally deliver the long-sought goal t
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of noninvasive detection of fetal aneuploidy. Recent data, however, indicate oa
that NGS might even be able to offer a much more detailed appraisal of the sh
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fetal genome, including paternal and maternal inheritance of point mutations md:
for mendelian disorders such as β-thalassaemia. Although these o
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developments are very exciting, in their current form they are still too complex hrbl
and costly, and will need to be simplified considerably for their optimal c
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translation to the clinic. In this regard, targeted NGS does appear to be a step la
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in the right direction, although this should be seen in the context of ongoing etr
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progress with the isolation of fetal cells and with proteomic screening markers. ft
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In the past few years it has become clear that Down Syndrome (DS) Cytogenetic Register over
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the demographic shift towards increasing the period 1989–2008 (Ref. 5), which reported a
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maternal age for pregnancies in developed 71% increase in the number of diagnosed cases i
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nations, with its associated risks of fetal with DS, with no comparable increase in birth a
chromosomal anomalies, will necessitate a rate. The increase in the number of cases n
change in current strategies for prenatal with DS was largely attributed to the mi
screening and detection of fetal aneuploidies concomitant increase in maternal age, as more
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(Refs 1, 2, 3, 4). This is illustrated by a recent than 20% of pregnancies now occur in mothers e
analysis of the English and Welsh National older than 35years (Ref.5). t
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1Laboratory for Prenatal Medicine, Department of Biomedicine / Department of Obstetrics and
Gynecology,University Hospital Basel, Switzerland.
2Department of Obstetrics and Gynecology,University Hospital Basel,Switzerland.
*Corresponding author: Sinuhe Hahn, Laboratory for Prenatal Medicine, Department of
Biomedicine/Department of Obstetrics and Gynecology, University Hospital Basel, Hebelstrasse
20,CH-4031 Basel, Switzerland. E-mail: [email protected]
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This development is likely to add additional although the number of reviews dealing with it
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costs to already strained healthcare budgets, the field has increased tremendously. Hence the m
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because positive cases from initial noninvasive scenario appears very similar to economic
screening based on ultrascans and maternal models of the ‘product life cycle’, where a Dn
serum biochemical analysis need to be verified particular item has progressed through e
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by invasive analysis such as amniocentesis or development, introduction and growth, and is ae
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chorionic villous sampling, which are labour- settling into a pattern of maturity and e
intensive examinations requiring highly skilled subsequent decline (Ref. 16). Although this fy
personnel (Ref. 6). To break this spiralling cost, simplistic view might lead to the impression mll
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it would be advantageous for any future that research in this field has waned, this is far
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noninvasive method to be so accurate that it from the truth, for this punctuated modicum in ri
could function as a ‘stand-alone’ test and not published reports indicates that the field has ff
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require further invasive verification. Naturally, it finally entered a phase where quality, using a
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will also need to be considerably cheaper than quantum leap in technological development,
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current invasive practices. An alternative rather than quantity is the prevailing trend. This ie
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scenario might be to use such a noninvasive test facetwill become veryevident in this review. al
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to reduce the cost and risk associated with the ra
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invasive analysis of the high number of false- The question of fetal cell-free DNA e
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positive cases resulting from current screening fragment size and concentration b
practice (Refs 7, 8). In either case, strenuous Itisnecessarytoreiteratethatthemajorproblem ae
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efforts will need to be undertaken in order to still hampering the use of fetal cf-DNA for the e
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ensure that cost-effective noninvasive prenatal noninvasive detection of fetal genetic loci that m
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diagnosis of DS cases finally becomes a reality are not distinct from maternal loci is that fetal
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(Ref. 8). sequences constitute only 5–10% of the total cf- sh
DNA in maternal plasma, and even less in o
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Has research in the field of noninvasive maternalserum (Refs 17,18, 19). md
prenatal diagnosis become ‘mature’? Groundbreaking research performed in the o
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Sincethediscoveryoffetalcell-freeDNA(cf-DNA) middle of the past decade on the biophysical r
inmaternalplasmaorserumin1997(Ref.9),more properties of cf-DNA indicated that fetal cf- hbl
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than 1000 papers have been published on this DNA was more fragmented and had a shorter
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topic. A cursory review of publications listed in size than comparable maternal cf-DNA la
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public depositories such as PubMed (http:// fragments (Refs 20, 21). Interestingly, this topic t
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www.ncbi.nlm.nih.gov/pubmed) suggests that still continues to be the focus of intense research e
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the most prolific period was probably the latter efforts and considerable debate (Refs 22, 23, 24). t
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half of the previous decade. In this period, The fragmentation was exploited for the om
several large-scale studies were initiated to test selective enrichment of fetal cf-DNA sequences
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the efficacy of this new-found tool for the (to up to 50% of total cf-DNA), thereby o
noninvasive determination of fetal genetic traits permitting the detection of otherwise masked i
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such as the fetal RhD (Rhesus D) gene or gender fetal genetic loci such as short tandem repeats, a
(Refs 10, 11, 12, 13). These data indicated that single-nucleotide polymorphisms (SNPs) and n
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this approach was indeed sufficiently robust that paternally inherited point mutations, such as m
widespread clinical application could be safely those involved in β-thalassaemia or
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implemented, and as a result concerted efforts achondroplasia (Refs 21, 25, 26, 27, 28). These e
were undertaken by multicentre consortia, such experiments, however, used conventional t
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as the European Union (EU)-funded SAFE agarose gel electrophoresis for the physical-size- D
Network, to standardise these assays (Refs 11, based separation of fetal and maternal cf-DNA
14). These data also indicated that the analysis species – a cumbersome, labour-intensive,
of cf-fetal DNA could form the secure and inefficient procedure that is prone to
sound basis for subsequent developments, such contamination. For the widespread applicability
as the noninvasive detection of fetal of this approach in the clinic, new approaches
aneuploidies (Ref.15). such as microfluidicswill be required.
In recent years this prolific output in Another key development during this period
publications seems to have largely tapered off, was verification of the long-standing suspicion
2
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thatfetalcf-DNAwasofplacentalorigin,andwas the study, because they failed to meet this it
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nottheresultofthedemiseoftraffickingfetalcells. important criterion. Furthermore, the approach m
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This was confirmed by a number of different makes a fundamental assumption concerning
approaches, such as analysis of (1) placental the underlying biology for it to work Dn
mosaicism, where key fetal loci such as the Y effectively – namely, that the interrogated alleles e
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chromosome were missing both in the placenta are transcribed at exactly the same rate. This ae
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and in fetal cf-DNA, but not in the male fetal might not be the case in many instances e
karyotype (Ref. 29), (2) molar pregnancies, (Refs 36, 37), which would make subsequent fy
where cf-DNA specific for the molar karyotype analysisunreliable. mll
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(46 XY) could be detected (Ref. 30), and (3) Although this RNA-based approach was
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epigenetic markers specific for placental tissues, subsequently explored by Sequenom, Inc., USA, ri
such as the hypomethylated maspin (SERPINB5) it appears not to have been successful. ff
or the hypermethylated RASSF1 genetic loci Unfortunately, details of this study have not se
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(Refs 31,32, 33). been divulged; it would have been interesting to
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The latter markers could prove to be useful as determine the cause for its apparent failure, ie
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gender-independent tools to verify the presence which perhaps could have been rectified in al
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of fetal cf-DNA in ambiguous diagnostic cases, subsequent studies. ra
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such as when determining fetal gender or RhD e
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status (Ref. 10). It might also be possible to use Epigenetic allelic ratios b
these as tools to quantify fetal cf-DNA levels A second indirectapproach, which was explored ae
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(Ref. 34). Reliable, accurate assessment of fetal around the same time, involved epigenetic e
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cf-DNA levels, however, appears to require the differences of methylation to distinguish m
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use of high-copy sequences, such as DYS14 on placental (fetal) genetic loci from maternal loci
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the Ychromosome (Ref. 18). (Refs 31, 32, 38, 39, 40). The first report, on the sh
maspin gene on chromosome 18 (Ref. 38), again o
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Indirect methods for the noninvasive used heterozygous SNP alleles to determine md
detection of fetal aneuploidy using chromosomal dosage; in this study, however, no o
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cell-free nucleic acids clear distinction could be discerned between ro
Allelic transcript ratios cases with trisomy 18 and unaffected healthy hbl
Thefirstindicationthatcell-freefetalnucleicacids controls, even when using ‘pure’ fetal and c
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could be used for the noninvasive prenatal maternal genetic material, such as fetal material la
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detection of DS, in 2007, came from an indirect obtained by amniocentesis. Subsequently, t
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approach in which chromosomal dosage was however, the epigenetic approach has been e
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inferred from allelic gene transcript copy explored successfully for cases with trisomy 21 t
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numbers (Ref. 35). The study focused on the and trisomy 18 (Edwards syndrome; ES) om
gene PLAC4 (placenta-specific 4), located on (Refs 39, 40). In these recent studies much more
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chromosome 21, which was specifically encouraging results were obtained, in that all o
transcribed in the placenta but not in any five DS cases and eight out of nine cases with i
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maternal tissues. In this manner, the analysis of ES were correctly identified by the analysis of a
this gene product would be similar to that of the epigenetic markers in plasma DNA. The efficacy n
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Y chromosome, in that it would not be hindered of these assays might be improved by the m
by maternal background. To assess the dosage inclusion of further candidate epigenetic
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of each allelic transcript, heterozygous SNP loci biomarkers (Refs 39, 40). e
were used. These could then be quantitatively Inaveryrecentreport,ithasbeendemonstrated t
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assessed using mass spectrometry. In the thattheenrichmentoffetalcf-DNAfragmentsby D
analysis of samples from 10 DS cases and 56 methylated DNA immunoprecipitation can be
healthy controls, DS cases could be detected used for the successful determination of
withasensitivityof90%andaspecificityof96.5%. chromosome 21 ploidy (Ref. 41). In this study,
However, a serious caveat of this approach is hypermethylated fetal cf-DNA fragments,
that the SNP in PLAC4 needs to be identified in a previous study (Ref. 42), were
heterozygous in order to derive a conclusion enriched by immunoprecipitation and then
concerning chromosomal dosage. Hence, a very examined by conventional real-time quantitative
large number of cases had to be excluded from polymerase chain reaction (PCR). The fetal-
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specific DNA methylation ratio for each sample minute individual reactions, with each it
e
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was then calculated by comparing the sample individual reaction containing at most a single m
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CT (cycle threshold) value with the median CT template copy. A quantitative assessment of the
value of a pool of normal control cases. In those concentration of input template in the sample Dn
cases where the fetus had a normal karyotype examined is then made by counting the number e
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this ratio was determined to be of the order of individual positive PCR reactions(Fig. 1). ae
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of 1, whereas in cases with trisomy 21 this ratio This procedure has been demonstrated to e
was larger than 1. To increase the accuracy of permit a much more accurate quantification fy
this system, eight methylated genetic regions on than moreconventional approaches such as real- mll
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chromosome 21 were examined in parallel. In time PCR, but, more importantly, it permits the
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a blinded analysis, 14 cases with trisomy 21 detection of very small changes in input DNA. ri
could be correctly distinguished from 26 Thus, ‘digital PCR’ could perhaps indicate ff
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normal cases. The advantage of this system isthat whether a fetus was affected by DS, simply by
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itdoesnotrequireanyspecialisedequipment,and counting the number of chromosome-21-specific
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can readily be performed by the instruments target sequences in comparison to a similar ie
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currently present in most routine diagnostic locus on an unaffected chromosome (Refs 46, 47, al
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laboratories. 48, 49)(Fig.1). ra
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Astrategyinwhichtheamyloidgenelocus on e
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DigitalPCR:firsthintatdirectnoninvasive chromosome21andtheGAPDH(glyceraldehyde b
aneuploidy detection 3-phosphate dehydrogenase) gene locus on ae
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Mostofthestudiesexaminingfetalcf-DNAtodate chromosome 12 were co-examined by digital e
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have used a form of real-time PCR and Y- PCR was shown to be able to detect DS when m
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chromosome-specific sequences (Refs 17, 43). examining pure fetal genetic material obtained
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These studies indicated that the amount of by invasive means (Ref. 47) (Fig. 1). The report sh
fetal cf-DNA increases during gestation, and indicated that the method might also be suitable o
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disappears rapidly from the maternal circulation for the analysis of cf-DNA, in that ‘DS cases’ md
following delivery. Increases were also observed could be detected using artificial mixtures o
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in several pregnancy-related disorders or involving only 10% DS material, which is very r
conditions such as preeclampsia, preterm similar to the concentration of fetal cf-DNA in hbl
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delivery and trisomy 21, suggesting that this maternal plasma. A crucial limitation of these
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phenomenon could serve as the basis for a new observations, however, was that they would la
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generation ofscreeningtests. hold true only when 10000 or more individual t
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During this period, studies had indicated that PCR reactions weremonitored. e
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real-time PCR could be used for the rapid t
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determination of chromosomal ploidy on pure Next-generation sequencing and the om
fetal genetic material obtained by invasive possible advent of noninvasive DS
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means (Ref. 44). Because real-time PCR is not detection o
well suited for the detection of less than twofold Although reports from two research groups i
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differences in template copy numbers, special indicated that digital PCR might offer success a
conditions had to be introduced to detect the for the noninvasive detection of DS by the n
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1.5-fold difference in template concentration analysis of cf-DNA (Refs 47, 48, 49), these m
occurring in trisomy 21. It was, however, very studies also made it clear that these analyses
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clear that this approach would not be suited for would be conducted at the limits of current e
the noninvasive determination of DS, because of digital PCR platforms, which at the time offered t
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the overwhelming presence of maternal cf-DNA some 12000 individual reaction events. Hence, if D
fragments. this strategy were to be pursued, then its
Consequently, a different tackhad to be taken, successful transition would require an even
which was provided by ‘digital PCR’ (Ref. 45) greater level of ‘individual event analysis’ to
and the advent of microfluidic devices (Ref. 46). permit the necessary degree of discrimination
Unlike real-time PCR, where an ‘analogue’ between normal and DS cases.
signal of the entire PCR reaction containing the This was offered by the advent of ‘next-
entire input template is obtained, in ‘digital generation sequencing (NGS)’, also termed
PCR’the PCR reaction is split into thousands of ‘deep sequencing’, whereby the entire genomic
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Euploid fetus Aneuploid fetus A
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12:12 12:12 Chromosomal Dn
21:21 21:21:21 complement e
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PCR for chromosomes 12 and 21
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Digital PCR readout on
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Ratio 21:12 = 1.0 Ratio 21:12 = 1.5
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Detection of fetal aneuploidy using digital PCR on
Expert Reviews in Molecular Medicine © 2011 Cambridge University Press tie
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Figure1.DetectionoffetalaneuploidyusingdigitalPCR.Inthisprocedure,fluorescentPCRspecificfor rh
e
sequenceson chromosomes12and 21is carried out inindividual microreaction chambers.The amount of c
input template is titrated in such a fashion that each microreaction vessel contains >1 copy. After the b
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plateauphasehasbeenreached(approximately40cycles),thePCRreactionisterminated,andthenumber
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of positive reactions for each locus is counted. In euploid cases the ratio of blue (chromosome 12) to red e
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(chromosome 21) signals should be 1, whereas in cases with Down syndrome the ratio of blue m
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(chromosome 12) to red (chromosome 21) signals should be 1.5 (illustrated in more detail in Ref. 46). This
oa
method hasto date not been successfully used forthe detection offetal aneuploidy using cell-free DNA in
sh
maternalplasma.
o
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md
o
o
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templateisfragmented,sequencedinshortreads number of chromosome 21 reads could lead r
and then reassembled through complex to an unparalleled discrimination between hbl
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bioinformatic comparison with a genomic pregnancies bearing a fetus with trisomy 21 or
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database (Ref. 50). In this manner, subtle those with a normal karyotype (Refs 53, 55). la
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mutations having key roles in tumour initiation Furthermore, because this analysis was not t
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andprogressioncouldbeascertained(Refs51,52). restricted to chromosome 21, it was also possible e
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Apartfromofferingunprecedenteddetailwith to detect twocases with ES (trisomy 18) and one t
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regardtogenomicalterations,theNGSapproach case with Patau syndrome (trisomy 13), thereby om
alsoofferedauniqueopportunitytoovercomethe indicating the possible widespread applicability
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current limitation of digital PCR, by providing of thistechnology (Ref.55). o
information concerning tens of thousands of The downside of this approach is the i
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sequence reads per chromosome; more than prohibitively high cost per sample and the a
60000 reads might be recorded for chromosome length of time taken for sample preparation, n
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21,and many millions over the entiregenome. sequencing and subsequent bioinformatic m
InthecaseofaDSfetus,therewouldbeasmall analysis, which occupies the better part of
r
increase in the number of chromosome 21 reads, severaldaysper sample (Ref.56). e
whereas there should not be any comparable t
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alteration across the other chomosomes. As Is NGS ready for clinical application? D
such, in a manner akin to digital PCR, by Neverthless, two very recent publications have
simply counting the number of chromosome suggested that the NGS approach might be
21 reads, and then comparing these to a much ready to make the transition from the research
larger number of sequence reads over the entire laboratory to clinical routine (Refs 7, 8, 34). In
genome, it should be possible to determine the the first of these studies, 753 pregnant women at
ploidy of chromosome 21 (Fig. 2). This indeed risk of having a fetus affected by DS, and who
was the case, and it was clearly demonstrated were therefore about to undergo an invasive
that even minute quantitative alterations in the prenatal diagnostic procedure, were recruited
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it
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A
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Cell-free tb
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DNA in plasma
fy
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Isolate cell-free DNA fragments and prepare library on
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Examine by next-generation sequencing al
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ATG, TTA, e
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CAT, ATT,
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Enumerate sequences/chromosome
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Compare with reference sample pool to determine ploidy an
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Detection of fetal aneuploidy using next-generation sequencing
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Expert Reviews in Molecular Medicine © 2011 Cambridge University Press
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Figure2.Detectionoffetalaneuploidyusingnext-generationsequencing.Inthisprocedurethecell-free t
DNAfragmentsinmaternalplasmaareisolated,andalibrarywithspecialsequencetagsisthenmade.These e
tags permit subsequent multiplex analysis. The library is examined by next-generation sequencing, which D
determines the sequence of each and every fragment. By bioinformatic analysis these sequences are
ascribedtochromosomallocations.Followingthis,thenumberofsequencereadsforeachchromosomeis
counted. For chromosome 21 this is typically of the order of several thousand reads, which can then be
comparedwith several millionreadsspreadacrossthegenome.Ifthefetusis affectedbyDownsyndrome,
thenslightlymorereadswillberecordedforchromosome21comparedwiththosefromaeuploidfetus.By
comparing these data with a bank of reference samples, and by the use of predetermined cut-off
values (Z score), the ploidy of the sample being examined can be determined (described in more detail in
Refs53,54).
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(Ref. 7); of these, full karyotyping determined Can NGS be simplified? it
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that 86 had afetus affected by DS. To reduce the The above results have indicated that the use of m
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complexity and cost of the NGS analysis, a complex cutting-edge genomic tools such as
multiplex approach was tested, whereby eight NGS can finally offer the long-sought dream of Dn
samples were examined in parallel (8-plex). This noninvasive detection of fetal aneuploidy e
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was compared with a more costly duplex (Refs 1, 56). However, this comes at a very high ae
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analysis on 314 samples (two samples examined cost and unacceptably long analysis. A possible e
simultaneously). The 8-plex analysis yielded a strategy to overcome these disadvantages is the fy
sensitivity of 79.1% and a specificity of 98.9%, targeting of only those chromosomes or regions mll
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whereas the duplex assay yielded 100% of interest such as those on chromosome 21 for
on
sensitivity and 97.9% specificity (Ref. 7). To the detection of DS cases. Although several ri
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make the assay more efficient, a cohort of different strategies exist that permit some form
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normal samples was used as a standard, of target enrichment prior to the subsequent
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permitting a threshold cut-off value (termed Z) sequencing step, they have to date been limited
on
to be determined: values above this were to examination of intact genomic DNA samples, ie
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deemed toshowDS cases (Fig. 2). and not fragmented DNA species such as those al
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Although it is unfortunate that the duplex found in cf-DNA (Ref. 57). Furthermore, these ra
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analysis was determined to be superior to the procedures, such as on-array capture, require e
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less complex 8-plex analysis, it is not entirely rather large concentrations of input template b
surprising, in that for the 8-plex assay only DNA (up to 7.5μg per sample). Hence, an ae
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approximately 300000 total reads were recorded approach that permitted targeted sequencing of e
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per sample. This translates to a small fraction of the small quantities of fragmented DNA in m
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the billions of reads required to access the entire maternalplasma had to be sought.
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genome, and furthermore implies that perhaps Very recently, a solution hybrid selection sh
less than 10000 chromosome-21-specific reads method using ultralong oligonucleotides, o
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were recorded. Because the NGS assay, like its commercially marketed by Agilent, USA, has md
digital PCR predecessor, relies simply on the been used for targeted enrichment (Ref. 57) o
o
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counting of individual events, it would seem (Fig. 3). A capture library specific for the X r
that here simply too few single events were chromosome was hybridised with 500ng of an hbl
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being interrogated. amplified plasma DNA library, and the selected
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In the second, independent study, 449 samples capture targets were pulled down using a la
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were examined by a more detailed and robust combination of biotinylated oligonucleotide t
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pair-end 4-plex NGS procedure in which probes and streptavidin-coated magnetic beads. e
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approximately 5 million reads per sample were The enriched target DNA was then subjected to t
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obtained (Ref. 34). In this examination all 39 a further 12 rounds of PCR with specially om
cases with DS were correctly identified, tagged primers necessary for the subsequent
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although one normal case was incorrectly sequence analysis. In the examination of 12 o
classified as DS, thereby yielding 100% maternal plasma samples, this targeted capture i
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sensitivity and 99.7% specificity. An important procedure led to a mean enrichment of 213-fold. a
quality control in this study is that the This enrichment was also reflected in the n
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concentration of fetal cf-DNA was monitored, increased ability to detect fetal-specific loci on m
and samples with less than 3.9% were excluded the X chromosome, which changed from 3.5% in
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(Ref. 34). the un-enriched samples to 95.9% in the samples e
EveniftheNGSassaysareconsiderednottobe subjected to atargeted enrichment step. t
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100%effective,andcouldnotbeusedasastand- Aconcernwithanyenrichmentapproachisthat D
alonediagnostictestforDS,ithasbeensuggested itmightleadtobias,bypreferentialenhancement
that their use as a secondary-tier screen of high- of select sequences, including the preferential
risk cases could lead to a 98% reduction in the accumulation of maternal cf-DNA sequences
number of invasive procedures (Ref. 7). As such, over fetal ones. However, in this study the
this avenue certainly does appear to be an proportion of fetal to maternal sequences was
attractive route to pursue (Ref. 8). Accordingly, similar in un-enriched loci (∼16–30% from
large-scale clinical studies are being launched in first to third trimester) and in enriched X-
Europe and elsewhere. chromosome regions (∼15–32%) (Ref. 57). These
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results are encouraging, because they could it
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facilitate targeted enrichment of more crucial m
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fetal loci, such as those on chromosome 21,
thereby paving the way for a less complex Dn
noninvasive test for DS. e
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Cell-free ae
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How much information is available from
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The firstseries ofexperimentswithNGS focused a
Isolate cell-free DNA fragments onselectchromosomesandcomparedthenumber on
and prepare library ofreadsobtainedforthesetothoseobtainedforthe frfi
entire genomic complement. Because close to se
65000 individual loci are counted on ng
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chromosome 21 alone, it was open to debate
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whether this approach would be sufficiently t
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Hybridise to SureSelectTM sensitive to detect more subtle chromosomal ral
Oligo Capture Library aberrations such as the Robertsonian rh
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translocation between chromosomes 21 and 15 c
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(or 14), which has a role in 2–3% of cases with ae
DS (Ref. 1). As the Down Syndrome Critical h
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Region associated with this translocation is a t
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lot smaller than the entire chromosome 21 s
commonly involved in DS, it was unclear oa
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whether the NGS approach would be able to
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detect this alteration (Ref. 1). :
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Select bound DNA fragments However, this view has been altered bya new o
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landmark publication, in which the entire cf- o
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DNA present in a maternal plasma sample was hbl
sequenced (Ref. 24). The data indicated that the c
l
entire fetal genome complement is present in la
cf-DNA in maternal plasma, and that this can be an
t
mined to show minute details such as mutations er
e
f
t
fa
Examine by next-generation o
m
sequencing Figure3.Schematicrepresentationofatargeted
sequencing approach using the SureSelectTM n
Target Enrichment System. In this procedure the o
ATG, TTA, i
cell-free DNA fragments are isolated and a library t
a
CAT, ATT, is generated as per the standard next-generation
n
sequencing protocol. Prior to sequencing,
TCA, TAG however, this library is hybridised to the mi
SureSelectTM Oligo Capture Library, which is
r
Schematic representation of a
manufactured in such a manner that it will e
targeted sequencing approach using recognise a specific chromosome, such as t
e
the SureSelectTM Target Enrichment chromosome 21. These oligo sequences contain D
magnetic particles to permit their retrieval in a
System
magnetic field. Hence, following hybridisation
(65°C, 24h), captured sequences are selected by
Expert Reviews in Molecular Medicine
magneticselection,andunselectedsequencesare
© 2011 Cambridge University Press
washed away. The bound fragments are then
purified and prepared for sequencing and
Figure3.Schematicrepresentationofatargeted examined as described for Figure 2. This
sequencing approach using the SureSelectTM procedurewasrecentlyshowntopermita213-fold
TargetEnrichmentSystem. enrichmentofthetargetedXchromosome(Ref.57).
8
Accessioninformation:doi:10.1017/S1462399411001852;Vol.13;e16;May2011
©CambridgeUniversityPress2011.Re-usepermittedunderaCreativeCommonsLicence–by-nc-sa.
reviews
expert
http://www.expertreviews.org/ in molecular medicine
n?
involvedinβ-thalassaemia;surprisingly,thestudy unclear how quickly these developments will be it
e
A
correctlydiscernedthatthefetushadinheritedthe translatable into the clinic. m
paternalcodon41/42mutation,involvingaCTTT For this reason there has been a resurgent N
deletion,butnotthematernal−28A→Gmutation. interest in the isolation of fetal cells from Dn
Although this analysis was very complex, maternal blood. This have been fostered by e
l
involving almost 4 billion reads and 900000 the development of (1) sophisticated high- ae
tb
SNPs, it is a striking indicator of what might lie throughput automated scanning devices, which e
ahead – namely, the ability to obtain a full fetal permit the rapid identification of putative target fy
karyotype down to miniscule single-nucleotide fetal cells among a large pool of maternal cells mll
a
detail from a single maternal blood sample (Refs 61, 62), and (2) efficient microfluidic
on
(Ref. 24). systems and electronic micromanipulation ri
ff
systems, which permit the effective retrieval of
se
individual traffickingfetal cells(Refs 59,63, 64).
The question of intellectual property and ng
Eventhoughthisprocesswillmostlikelyalsobe
how to optimise procedures on
laborious,ithasseveraladvantagesovercf-DNA ie
An important concern that needs to be t
analysis, in that fetal cells offer a pure source of al
addressed is how intellectual property and l
fetal genomic material. Furthermore, widely ra
commercialisation will affect future research and rh
standardised procedures commonly used in e
applications (Ref. 58). For the best possible c
many diagnostic laboratories, such as FISH b
service to reach the patient, clarity is essential;
(fluorescence in situ hybridisation), can be ae
otherwise, conflicting reports and views might h
applied for the very rapid detection of e
prevent superior products from being t
chromosomal anomalies (Ref. 65). In addition, m
developed. In the case of fetal cf-DNA, such an s
use can now be made of a wide body of
issue was raised by the commercialisation of a oa
noninvasive test for fetal RhD determination in experience in the analysis of single cells, a sh
routine practice in many in vitro fertilisation o
the EU, and subsequently in the USA (Ref. 58). :
clinics offering preimplantation genetic md
Although it was clear to leading researchers in
diagnosis (PGD) (Ref.66). o
the field that the tests initially marketed might oo
A possible further advantage is that the r
be flawed or were not state of the art, it was felt intellectual property situation in this area is not hbl
that it would be a weary process to convince the c
as complex as that of cf-DNA, in that many of
commercial parties involved to change their the original patents are close to expiry, and no lal
standard of practice and to adopt more modern an
commercial entity holds a wide portfolio of key t
effective approaches. This issue was further er
elements. As such, it will be of interest to e
highlighted by the near-simultaneous reports f
monitor progress in this sphere of research, t
concerning the application of digital PCR or fa
becauseitmightyethaveafewsurprisesinstore. om
NGS, leading to conflicting reports in the lay
media astowho the main patent claimants were n
The challenge of proteomics
o
(Refs 1, 59). It is currently also unclear what
The development of the first-trimester DS i
business model or strategies should be used in t
screening test using acombination of ultrasound a
translating this research from the bench to the
measurement of nuchal translucency, serum n
clinic. Hence, it might be worthwhile to echo i
analyte analysis and maternal age has led to a m
previous concerns that such uncertainty might
dramatic increase in the ability to detect
hinder or restrict further research in the field r
pregnancies at-risk of bearing an aneuploid e
(Ref. 60).
fetus (85% sensitivity) when compared with t
e
the previous generation of second-trimester D
Are fetal cells in maternal blood still screening tests (65% sensitivity), which relied
worth pursuing? solely on serum analyte analysis in combination
Despitetheenormousstridesthathavebeenmade with maternal age (Ref. 67). This facet is
with the determination of fetal aneuploidies reflected in the report on the English and Welsh
through the use of cf-DNA, the latest reports National Down Syndrome Cytogenetic Register
have indicated that these will be very labour analysis, which indicated that the vast majority
intensive, and require extremely high-tech of DS cases were detected by current screening
devices and bioinformatic services. Hence it is practice (Ref. 5).
9
Accessioninformation:doi:10.1017/S1462399411001852;Vol.13;e16;May2011
©CambridgeUniversityPress2011.Re-usepermittedunderaCreativeCommonsLicence–by-nc-sa.
reviews
expert
http://www.expertreviews.org/ in molecular medicine
n?
The downside of this startling development is clinical service. Although the isolation and it
e
A
that the first-trimester screen is still hampered analysis of trafficking fetal cells might remain a m
by a rather high false-positive rate of 5–8% peripheral test, it could be useful in those N
(Refs 68, 69). This implies that a large number of centres already offering FISH or PGD services. Dn
healthy pregnancies are being subjected to Should multiparameter mass spectroscopy and e
l
unnecessary invasive procedures – a healthcare quantitative proteomics finally come of age, it ae
tb
risk for mother and unborn child, as well as a might yield a set of biomarkers with e
considerable financial burden for healthcare unparalleled specificity, not only for DS fy
services. Furthermore, almost 15% of cases screening but also for other pregnancy-related mll
a
remain undetected and, because these occur in disorders such aspreeclampsia.
on
the group of pregnant women not judged to be ri
at a higher risk (<35 years old), could result in Acknowledgements and funding ff
se
undesiredlivebirths. This report was funded by the clinical research
ng
Therefore, increasing the efficiency of this fund of the University Women’s Hospital Basel.
on
test has been proposed, perhaps by the addition The authors thank the referees for their critical ie
t
of further biochemical analytes, such as and insightfulcomments. al
l
members of the activin family (Refs 70, 71). The ra
rh
inclusion of further highly specific blood-based References e
c
analytes might be possible as a result of the 1 Hahn,S.,Jackson,L.G.andZimmermann,B.G. b
development of quantitative proteomic (2010)Prenataldiagnosisoffetalaneuploidies: ae
h
technologies as part of the Human Proteome post-genomicdevelopments.GenomeMedicine e
t
Association, especially the affiliated plasma 2,50 m
s
proteome project (Ref. 72). The placenta in DS 2 Bianchi,D.W.(2010)FromMichaeltomicroarrays: oa
cases exhibits structural alterations, which are 30yearsofstudyingfetalcellsandnucleicacidsin sh
most likely the result of alterations in protein maternalblood.PrenatalDiagnosis30,622-623 o
:
expression, and so it is possible that these 3 Ferguson-Smith,M.A.andBianchi,D.W.(2010) md
changes will be reflected in the maternal plasma Prenataldiagnosis:past,present,andfuture. oo
o
proteome (Ref. 73). In this regard, the use of PrenatalDiagnosis30,601-604 r
quantitative isobaric labelling has been shown to 4 Lo,Y.M.(2010)Noninvasiveprenataldiagnosisin hbl
c
permit the detection of significant alterations in 2020.PrenatalDiagnosis30,702-703 l
the levels of several placenta-derived peptides in 5 Morris,J.K.andAlberman,E.(2009)Trendsin alna
DS cases when comparedwith controls (Ref. 74). Down’ssyndromelivebirthsandantenatal t
er
Analogous observations have been made by diagnosesinEnglandandWalesfrom1989to2008: e
f
other research groups using a variety of analysisofdatafromtheNationalDownSyndrome t
fa
different proteomic approaches (Refs 75, 76, 77, CytogeneticRegister.BritishMedicalJournal339, om
78). It now remains to explore how useful these b3794 n
potential biomarkers will be in increasing the 6 Philip,J.etal.(2004)Latefirst-trimesterinvasive o
detectionrateofthecurrentfirst-trimesterscreen. prenataldiagnosis:resultsofaninternational i
t
randomizedtrial.ObstetricsandGynecology103, a
Summary and conclusion 1164-1173 n
i
Tremendous strides have been made with the 7 Chiu,R.W.etal.(2011)Non-invasiveprenatal m
analysis of fetal cf-DNA in maternal blood, in assessmentoftrisomy21bymultiplexedmaternal
r
that we now stand on the threshold of a new plasmaDNAsequencing:largescalevaliditystudy. e
generation of noninvasive diagnostic tools, BritishMedicalJournal342,c7401 t
e
which might even permit a detailed analysis of 8 Jackson,L.andPyeritz,R.E.(2011)Molecular D
the entire fetal genome or a full karyotype. technologiesopennewclinicalgeneticvistas.Science
However, it remains to be seen how rapidly TranslationalMedicine3,65–62
these can be translated into the clinic, in a 9 Lo,Y.M.etal.(1997)PresenceoffetalDNAin
manner that is cost effective and accessible to maternalplasmaandserum.Lancet350,485-487
all. In this context it will be interesting to see 10 vanderSchoot,C.E.,Hahn,S.andChitty,L.S.(2008)
which system, namely NGS, digital PCR Non-invasiveprenataldiagnosisanddetermination
or enrichment of methylated fetal cf-DNA offetalRhstatus.SeminarsinFetalandNeonatal
fragments, passes the final hurdle to enter Medicine13,63-68
10
Accessioninformation:doi:10.1017/S1462399411001852;Vol.13;e16;May2011
©CambridgeUniversityPress2011.Re-usepermittedunderaCreativeCommonsLicence–by-nc-sa.