NON INVASIVE PRENATAL DIAGNOSIS OF ANEUPLOIDY:
NEXT GENERATION SEQUENCING OR FETAL DNA
ENRICHMENT?
Webb A, Madgett TE, Miran T, Sillence K, Kaushik N, Kiernan M, Avent ND*
*Corresponding Author: Professor Neil D. Avent, School of Biomedical and Biological Sciences, Faculty of
Science and Technology, A411 Portland Square, Drake Circus, Plymouth, Devon, PL4 8AA, UK; Tel.: +44-
(0)1752-584884; Fax: +44-(0)1752-584605; E-mail: neil.avent@plymouth.ac.uk
page: 17
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METHODS TO DETECT FETAL ANEUPLOIDY BY NON INVASIVE PRENATAL DIAGNOSIS
Methods Based on Allelic Ratio: RNA-Single
Nucleotide Polymorphism Allelic Ratio Approach.
The presence of free fetal RNA (ffRNA) in maternal
plasma was established in 2000 by Poon et al. [22]
who showed that Y chromosome-specific zinc finger
protein (ZFY) mRNA could be found in the plasma
of women carrying a male fetus. Further studies
showed that ffRNA is surprisingly stable [23] and
present in maternal plasma as early as 4 weeks gestation
[24]. As different tissues express their own
individual mRNA profiles, it follows that some
mRNAs may be placenta-specific and therefore fetal specific. Ng et al. [25] showed that placenta specific
mRNAs could be detected in maternal plasma,
and in 2007, a placental-specific mRNA transcribed
from PLAC4 on chromosome 21 was discovered
[26]; use of these could circumvent the problems
caused by maternal background DNA.
Lo et al. [26] developed the RNA-SNP (single
nucleotide polymorphism) allelic ratio approach.
This technique exploits SNPs that cause sequence
variation between alleles. A heterozygous euploid
fetus should yield equal proportions of each allele,
giving an allelic ratio of 1:1. However, a heterozygous
triploid fetus would yield the allelic ratio 1:2
or 2:1 [27]. Lo et al. [26] used reverse transcription
(RT)-PCR to amplify a PLAC4 SNP containing locus,
followed by primer extension. Mass spectrometry
was then used to determine allelic ratio (Figure
1). Maternal plasma of 57 women carrying euploid
fetuses and 10 carrying T21 fetuses was analyzed.
The method had a sensitivity of 90.0% and specificity
of 96.0% [26]. Larger trials to refine the normal
reference ranges could potentially improve sensitivity
and specificity [27]. Regrettably, RNA-SNP allelic
ratio methods are limited to fetuses heterozygous for
the SNP under analysis. Among 119 placentas genotyped
by Lo et al. [26] for the most common PLAC4
SNP (rs8130833), 42.0% were homozygous and the
method would have therefore been uninformative. The Digital RNA-Single Nucleotide
Polymorphism Allelic Ratio Approach. The digital
RNA-SNP allelic ratio approach is an adaptation
to the RNA-SNP allelic ratio method made to utilize
the sensitivity of digital PCR [28]. Rather than one
reaction mix, digital PCR involves thousands of reactions
running in parallel. The template strand solution
is diluted so that a maximum of one template
strand is added to each reaction. Lo et al. [28] used
384-well plates for digital ReTi-PCR of the PLAC4
SNP, rs8130833. Uninformative wells (i.e., those
containing no or multiple signals) were discounted
and wells containing A or G PLAC4 allele were
counted and their ratio calculated. Using maternal
plasma RNA samples, four T21 fetuses and nine euploid
were correctly identified. However, although
digital PCR is more sensitive than ReTi-PCR, the
digital RNA-SNP allelic ratio approach it is still
limited to heterozygous fetuses. The methodology
entered commercial trials under the governance of
Sequenome Inc. (San Francisco, CA, USA) but was
subsequently found to be an unreliable technology
and unlikely to see routine application in NIPD.
Epigenetic Allelic Ratio Approach. The epigenetic
allelic ratio (EAR) approach is similar to
the RNA-SNP allelic ratio approach but rather than
targeting fetal specific mRNA, it exploits epigenetic
phenomena that alter DNA expression without
altering sequence; the most well known of these is
cytosine methylation. Methylation patterns differ
between tissues; genes that are differentially methylated
between mother and fetus have been identified
allowing an opportunity to selectively target
fetal-specific DNA with the use of methylation specific
primers [22,27].
Chim et al. [29] demonstrated that the maspin
gene (SERPINB5) promoter is unmethylated
(U-mapsin) in the placenta but hypermethylated
in maternal blood cells. SERPINB5 is located at
18q21.33 [30], providing the opportunity for an
EAR-based approach to T18 detection. Tong et al.
[30] used the EAR approach, first using bisulphite
conversion followed by methylation-specific PCR
and primer extension reactions designed to distinguish
the A and C allele of U-mapsin based on size
[30]. The test had a sensitivity of 100.0% but had a
9.7% false positive rate [30]. Theoretical modelling
suggested that 200 sequence copies were needed at
the start of PCR to achieve diagnostic sensitivity and specificity of 97.0%. However, bisulphite conversion
has been reported to cause DNA degradation
of up to 96.0% [30]. Taking this into account, Tong
et al. [30] predicted that only 20 sequence copies
would have remained in their samples after bisulphite
conversion due to low blood volume used; this
could explain the high false positive rate.
Although the EAR approach was successfully
demonstrated, strategies need to be developed to
overcome DNA degradation by bisulphite conversion.
Weber et al. [31] described a way of enriching
methylated DNA using immunoprecipitation with
the use of an antibody against 5-methylcytosine;
methylated DNA immunoprecipitation (MeDiP)
has since been used to successfully enrich fetal hypermethylated
DNA [32]. Alternatively, degradation
of maternal unmethylated DNA by bisulphite
conversion would be acceptable so targeting genes
hypermethylated in the placenta but hypomethylated
in maternal tissues could be a solution [3].
Despite various groups showing methods based
on allelic ratio to work successfully, all are limited
to heterozygous fetuses. However, an allelic ratio
method could still have potential for use in practice
if multiple SNPs, with a combined heterozygosity
rate high enough to cover the general population,
were analyzed [27]. Allelic ratio methods also assume
both alleles are transcribed at an equal rate,
which is not always true [4], meaning analysis of
transcript ratios could be unreliable. However, successful
demonstration of allelic ratio methods suggests
this is not problematic. Finally, although allelic
ratios may successfully identify trisomies they
would not detect monosomies.
Methods Based on Relative Chromosome
Dosage. Using microfluidics digital PCR, Lun et al.
[33] re-evaluated the percentage of cell free DNA
that was fetal in maternal plasma and found there
was a median 9.7% in the first trimester rising to
20.4% by the third trimester. These levels are higher
than previously thought, suggesting direct measurement
of fetal chromosome dose may be possible
without prior enrichment. Relative chromosome
dose approaches work on the principal that in a
normal fetus the ratio of two chromosomes should
be 2:2. Comparing an affected chromosome in a
triploid fetus would give the ratio 3:2. A number
of methods based on chromosome dose have been
reported and are described below. Epigenetic-Genetic and Epigenetic-Epigenetic
Chromosome Dosage Approaches. Tong et al. [34]
combined a methylation restricted digest to digest
hypomethylated DNA and microfluidics digital PCR
to measure HLCS on chromosome 21 (a hypermethylated
fetal marker), RASSF1A on chromosome 3
(a hypermethylated fetal marker) and ZFY on the
Y chromosome. The epigenetic-epigenetic chromosome
dosage approach comparing hypermethylated
HLCS and RASSF1A ratio showed too great an
overlap between euploid and T21 fetuses. However
the epigenetic-genetic chromosome dosage approach
comparing hypermethylated HLCS and ZFY
ratio in euploid and T21 fetuses was discriminative
but limited to male fetuses. Other groups have also
successfully used the EGG chromosome dose approach,
for example, Tsui et al. [35] identified T18
fetuses with a sensitivity of 88.9% and specificity
of 96.3%. However, Tsui et al. [35] also used ZFY
limiting use to male fetuses. ZFY is often used for
proof of principal studies but epigenetic-genetic
(EGG) chromosome dosage needs to be successfully
demonstrated with sex-independent markers
before it can be used in practice.
Digital Relative Chromosome Dosage Method.
There is a 1.5-fold increase in relative chromosome
dose in trisomies. Due to the exponential increase
in template strand number in ReTi-PCR, it is not
suited to detecting differences in template strand
concentrations below 2-fold [4,36,37]. Digital PCR
provides a more accurate measure of template strand
concentration.
Methodology involves the amplification of multiple
targets on different chromosomes in many thousands
of different PCR reactions (microfluidic PCR
or droplet PCR). In this example, two regions of the
human genome have been amplified, chromosomes
18 and 21. The relative abundance of the target DNA
is defined directly against one another, and thus a statistical
1:1 ratio of each chromosome would indicate
a euploid fetus and a (theoretical) 2:1 ratio (21:18)
would indicate aneuploidy. Despite the admix of fetal
and maternal DNA in maternal plasma, like next generation
sequencing (NGS) (see Figure 2), digital PCR
is able to differentiate such a difference, although
clearly the actual difference in ratio would not be 2:1
due to the presence of maternal DNA.
The digital relative chromosome dosage (RCD)
method directly targets a non polymorphic locus on the chromosome of interest and on a reference chromosome
without differentiating between maternal
and fetal DNA. Lo et al. [28] used digital PCR to
measure a non polymorphic locus on chromosome
21 and chromosome 1. The number of wells in
which there was a positive PCR for either locus was
counted and their relative dose calculated. Euploid
and T21 fetuses in artificial mixtures of placental
and maternal DNA were successfully identified but
the method was not tested using maternal plasma
from T21 pregnancies. However, despite RCD not
differentiating between maternal and fetal DNA,
computer modeling estimated 97.0% of fetuses
would be correctly identified by 7680 digital PCRs
in cases where the cffDNA constituted 25.0% of the
DNA [28]. Nevertheless, this is higher than previously
reported levels in maternal plasma. Moreover,
other groups have shown the digital RCD approach
to be feasible with fetal fractions of 10.0% [38] although
some level of enrichment may still first be
needed for use in the first trimester.
Next Generation Sequencing (NGS) or
Massively Parallel Sequencing. Massively parallel
sequencing (MPS) is a method through which
the entire genome can be sequenced using millions
of short sequence reads. These are then reassembled
by a computer program using genomic databases to
compare the reads to the known genomic sequence
[3,4,39]. A number of groups have demonstrated that if cffDNA is sequenced in this way and the sequence
reads assigned to each chromosome are then
counted, whether a chromosome is over- or underrepresented
can then be calculated [39,40]. This is
similar to the approach taken for the digital relative
chromosome dose method. However, NGS generates
far more sequence reads, possibly over 60,000
for chromosome 21 alone, suggesting it should be
more sensitive to small changes in genomic representation
[4]. The NGS could therefore have the
potential of identifying partial trisomies, although
further research is needed to investigate this.
Fan et al. [39] were able to identify nine T21 cases,
two T18 and one T13, distinguishing them from
six normal euploid cases, using shot gun sequencing
(Figure 2). However, the blood samples used in this
study were drawn 15 to 30 min. following an invasive
procedure, this may have influenced the results
by introducing additional fetal DNA into the maternal
circulation [3]. Furthermore, Fan et al. [39] argue
that using a digital PCR assay, they estimated fetal
DNA in their samples constituted <10.0% of total
cell free DNA, in line with previous studies.
More recent studies have taken into account
that differing GC contents between chromosomes
results in nonuniform sequencing by MPS and corrected
genomic representation to account for CG
content. By doing this, Chen et al. [40] found that
a specificity and sensitivity of 98.9 and 100.0%, respectively,
could be achieved for T13 detection and
98.0 and 91.9%, respectively for T18 detection.
In light of recently successful studies, Ehrich et
al. [41] conducted a blinded study on 449 plasma
samples from pregnant women (39 with T21 fetuses),
using the most up-to-date sequencing technology
available at the time, to investigate NGS for T21
diagnosis. Z-scores described in [42] were used to
standardize genomic representation and classify the
fetus as euploid or T21. The results were very successful
with a sensitivity of 100.0% and specificity
of 99.7%, suggesting that if the cost of MPS could
be reduced it could be used in practice. Recently,
using the single molecule sequencing technology
of Helicos (Cambridge, MA, USA) that does not
require a prior DNA amplification step, van den
Oever et al. [43] have demonstrated a greater sensitivity
of this platform above that of Illumina GAII
(San Diego, CA, USA) used in previous studies.
This may lead to utilization of NGS much earlier in NIPD, perhaps within the first trimester [44], as the
study demonstrated detection of trisomy 21.
All three relative chromosome dose methods
discussed are polymorphism-independent and have
successfully demonstrated the ability to detect trisomies.
Of the three relative chromosome dose methods,
NGS seems the most promising as, due to the
large amount of data it produces, it has high sensitivity
and specificity. It can also simultaneously
provide information on chromosome dose for all
chromosomes, and theoretically, has the potential to
detect partial trisomies and monosomies, although
this needs to be validated by further research.
Methods to Enrich Fetal DNA. One of the main
hindrances on NIPD is the dilution of fetal DNA in
maternal blood; this makes the quantitative nature of
aneuploidy diagnosis difficult. Therefore, a number
of methods have been investigated to enrich or prevent
the dilution of fetal DNA in maternal plasma.
Use of Formaldehyde. In 2004, Dhallan et
al. [45] hypothesized that a significant portion of
maternal DNA in maternal plasma is leaked from
maternal leukocytes following venipuncture due to
physical forces put on them during collection and
subsequent handling. Blood was collected from 69
pregnant women in tubes containing a 4.0% formaldehyde
neutralizing buffer. Analysis showed the
majority of samples had >25.0% fetal DNA and
27.5% had >50.0%, suggesting formaldehyde treatment
could successfully prevented leukocyte rupture.
However, no untreated blood was collected
in this trial so no comparisons between treated and
untreated samples could be made. Since 2004, a
number of groups have attempted to replicate the
enrichment of fetal DNA in maternal plasma using
formaldehyde without success [46,47].
Separation Based on Size by Gel Electrophoresis.
Another approach to fetal enrichment is
to separate cffDNA from maternal based on size.
This can be done by gel electrophoresis [48]. In early
pregnancy (13 + 2 weeks gestational ages), Li et
al. [48] found that 85.5% of fetal DNA is less than
0.3 kb. Fetal DNA also constituted 28.4% of the
<0.3 kb fraction in maternal plasma, increasing to
68.7% in the third trimester. A study by Chan et al.
[49] supports Li et al. [48], concluding that >99.0%
of fetal derived DNA is shorter than 313 bp. Gel
electrophoresis has since been used to successfully
enrich fetal DNA for detection of point mutation in b-thalassemia [50]. However, isolation of size fractions
by gel electrophoresis is considered too time
consuming and prone to contamination to allow its
widespread use [3].
Co-Amplification at Lower Denaturation
Temperature Polymerase Chain Reaction. In
2008, Li et al. [51] reported on (co-amplification
at lower denaturation temperature) COLD-PCR, a
variation of PCR that can selectively amplify minority
alleles from a background of wild-type alleles.
This technique works on the basis that even
a single nucleotide difference between the minority
and wild-type allele may lower the critical denaturation
temperature (Tc). If so, the minority allele
could be denatured at a lower temperature than
the wild-type sequence, allowing only the minority
allele sequence to bind with the primers and be
amplified [52]. Li et al. [51] used COLD-PCR to
identify mutations in a number of genes associated
with human cancer that had previously been missed
and suggested that COLD-PCR could also be used
for detection of “fetal alleles in maternal blood.”
However, in aneuploidies there may be no sequence
difference between the maternal and fetal allele of
interest. Moreover, it is possible that the shorter
length of cffDNA in comparison with maternal cell
free DNA may allow COLD-PCR to denature cffDNA
at a temperature at which maternal DNA would
remain double-stranded, allowing only cffDNA to
be amplified. There is currently no published study
on the enrichment of fetal DNA from maternal plasma
using this method but this could be a promising
technique. If fetal DNA could be enriched in this
way, not only could it aid detection of aneuploidies
but also aid detection of monogenic diseases where
a disease allele may have been maternally inherited.
Proteomics for New Down’s Syndrome Biomarkers
(for review, see [53,54]). Despite the
advances in molecular counting technologies, especially
NGS and digital PCR, better serum screening
biomarkers for Down’s syndrome and other conditions
are still an option, as NGS and digital PCR
methodologies are not yet applicable to mass-scale
screening. For this reason several large scale studies
were launched to analyze the maternal plasma proteome
in pregnancies where the mother was carrying
a Down’s syndrome fetus [53,55-60]. It is clear that a
number of overlapping biomarkers have been identified,
but the search for a truly differentiating trisomy 21 biomarker using this approach may still be a way
off, but may well be worth the effort expended.
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