REVIEW

CURRENT
OPINION Noninvasive prenatal diagnosis for single
gene disorders
Stephanie Allen, Elizabeth Young, and Benjamin Bowns

Purpose of review
Noninvasive prenatal diagnosis for single gene disorders is coming to fruition in its clinical utility. The
presence of cell-free DNA in maternal plasma has been recognized for many years, and a number of
applications have developed from this. Noninvasive prenatal diagnosis for single gene disorders has
lagged behind due to complexities of technology development, lack of investment and the need for
validation samples for rare disorders.
Recent findings
Publications are emerging demonstrating a variety of technical approaches and feasibility of clinical
application. Techniques for analysis of cell-free DNA including digital PCR, next-generation sequencing and
relative haplotype dosage have been used most often for assay development. Analysis of circulating fetal
cells in the maternal blood is still being investigated as a viable alternative and more recently transcervical
trophoblast cells. Studies exploring ethical and social issues are generally positive but raise concerns
around the routinization of prenatal testing.
Summary
Further work is necessary to make testing available to all patients with a pregnancy at risk of a single gene
disorder, and it remains to be seen if the development of more powerful technologies such as isolation and
analysis of single cells will shift the emphasis of noninvasive prenatal diagnosis. As testing becomes
possible for a wider range of conditions, more ethical questions will become relevant.
Keywords
cell-free fetal DNA, fetal cells, next-generation sequencing, prenatal diagnosis, single gene disorder

INTRODUCTION and the current state of play with regard to

The development of noninvasive prenatal diagnosis
(NIPD) for single gene disorders (SGDs) has been
slow in comparison with noninvasive prenatal test-
ing (NIPT) for aneuploidy and NIPD for fetal sexing
and fetal Rhesus status. This is largely due to the
complexity of testing required and the relatively
small numbers of patients with individual disorders,
some of whom have unique mutations. Rare dis-
orders are however collectively common. One in 17
people (7% of the population) will be affected by a
rare disease at some point in their lives [1], so there is
a clinical need to make NIPD testing available to this
patient group regardless of the investment required.
Since the discovery of cell-free fetal DNA
(cffDNA) in maternal plasma in 1997 [2], there
have been reports in the literature showing case
studies and proof of principle NIPD assays on a
number of SGDs on relatively small numbers of
patients (reviewed by Wong and Lo [3]). This review
updates on the more recent publications in this area,
clinical implementation.
CELL-FREE FETAL DNA
It is worth noting that fetal DNA can be retrieved
noninvasively from three sources – cffDNA in
maternal blood, circulating fetal cells in maternal
blood and transcervical trophoblastic cells. Most of
the published work to date focuses on circulating
cffDNA derived from placental trophoblasts and we
will therefore discuss this first. Some recent
West Midlands Regional Genetics Laboratory, Birmingham Women’s
Hospital NHS Foundation Trust, Birmingham, UK
Correspondence to Stephanie Allen, PhD, FRCPath, West Midlands
Regional Genetics Laboratory, Birmingham Women’s NHS Foundation
Trust, Birmingham, UK. Tel: +44 121 627 2710; fax: +44 121 627 2711;
e-mail: stephanie.allen@bwnft.nhs.uk
Curr Opin Obstet Gynecol 2017, 29:73–79
DOI:10.1097/GCO.0000000000000347

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Prenatal diagnosis

nucleotide polymorphisms (SNPs) into different infor-

KEY POINTS
 The majority of studies on NIPD of SGDs still involve
testing of cffDNA, although circulating fetal cells and
more recently transcervical trophoblast cells are being
explored as an alternative.
 Methods currently used include next-generation
sequencing, RHDO, digital PCR, COLD-PCR, real-time
PCR and minisequencing.
 Clinical implementation is ongoing in some places, but
is challenging due to the number and diversity of
disorders requiring validation of samples and
assay design.
 Studies investigating ethical and social issues are
generally positive but raise concerns such as the
routinization of testing, the associated increased
pressure to have prenatal testing and the potential
increase in terminations.
developments in the use of circulating fetal cells in
maternal blood and transcervical trophoblastic cells
will then be reviewed.
Table 1 lists recent publications that describe
techniques for NIPD of SGDs by analysis of cffDNA.
These are grouped by testing method, and we will
look in more detail into a few of the more com-
monly reported technologies.
HAPLOTYPE-BASED NEXT-GENERATION
SEQUENCING
An increasing number of studies are reporting the use
of haplotype-based analysis to determine the inher-
itance of parental mutations in a linkage type manner.
This approach requires the grouping of single
mative categories based on parental zygosity. Phasing
of parental SNP haplotypes is achieved by comparison
of genomic DNA samples from the mother, father and
affected proband in each family. Analysis of sequenc-
ing data from cell-free DNA (cfDNA) enables the
detection of the mutant or normal haplotype in the
fetus. Figure 1 shows the principle of this method
using maternally heterozygous SNPs in X-linked dis-
orders (Fig. 1a) and categories 3 and 4 SNPs in auto-
somal recessive disorders (Fig. 1b). Xu et al. [4]
performed NIPD on eight pregnancies at risk of the
X-linked recessive disorder Duchenne muscular dys-
trophy (DMD) in this way, using target enrichment
and massively parallel sequencing. Analysis of SNPs in
the maternal cfDNA using a Hidden Markov Model
correctly identified the high-risk or low-risk maternal
haplotype in all cases, as confirmed by conventional
prenatal diagnosis. Yoo et al. [5] carried out a similar
study with four DMD families, correctly identifying
one unaffected and three affected fetuses.
Other groups have reported the use of these
methods for the diagnosis of autosomal recessive
disorders. Chen et al. [6] expanded on their previous
work, in which they had developed haplotype-based
strategies for the NIPD of DMD and congenital deaf-
ness, to further investigate five families with preg-
nancies at risk of spinal muscular atrophy (SMA). The
analysis identified two unaffected pregnancies,
two carriers and one affected fetus, which were all
confirmed by invasive testing. Another report fol-
lowed a family with GJB2-associated hearing impair-
&
ment [7 ]. NIPD was combined with preimplantation
genetic diagnosis, NIPT for fetal aneuploidy and
invasive confirmation by amniocentesis. Although
this provided an effective management strategy for
the family, the cost of providing this combination of

Table 1. Techniques for analysis of cell-free fetal DNA for noninvasive prenatal diagnosis of single gene disorders

Technique Disorders Reference Number of reports

Haplotype-based NGS/relative Duchenne/Becker muscular dystrophy, spinal muscular [4–6,7 ,8 ,9–12] 9

& &

haplotype dosage atrophy, congenital adrenal hyperplasia, GJB2-associated

hearing impairment, Gaucher disease
Droplet digital PCR CF, neonatal diabetes and achondroplasia [13–15,16 ] 4
&

Minisequencing Achondroplasia [15] 1

Bespoke/targeted NGS Achondroplasia, thanatophoric dysplasia and lethal skeletal [17,18 ] 2
&

dysplasia
COLD-PCR and microarray CF, beta thalassemia [19] 1
Fragment analysis Huntington disease [20] 1
Allele-specific real-time PCR b-thalassemia [21,22] 2
cSMART Wilson disease [23] 1

Articles within the review period reporting NIPD of SGDs by analysis of cell-free fetal DNA. These are categorized into methods used for testing and disorders
tested. Source: original. CF, cystic fibrosis; COLD, coamplification at lower denaturation temperature; cSMART, ciruclating single-molecule amplification and
resequencing technology; NGS, next-generation sequencing; NIPD, noninvasive prenatal diagnosis; SGD, single gene disorder.

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 Noninvasive prenatal diagnosis for single gene disorders Allen et al.

FIGURE 1. Principles of relative haplotype dosage. (a) Relative haplotype dosage analysis for X-linked disorders showing how
over-representation of informative single nucleotide polymorphisms on the X chromosome is used to show whether the fetus has
inherited the mutant or normal copy of the gene. Adapted from [8 ] (open access). (b) Relative haplotype dosage analysis for
&

autosomal recessive disorders (original).

tests was considered prohibitively high in terms of single gene disorders study that was aimed at devel-
clinical application. oping tests with a level of affordability to enable
The haplotype-based approach was taken fur- clinical implementation. Two publications from the
ther during the noninvasive prenatal diagnosis for study detail the development of assays for NIPD of

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Prenatal diagnosis
Duchenne/Becker muscular dystrophy (DMD/BMD)
&
and SMA [8 ,9]. Methods were similar to those
above, except that analysis of cfDNA to determine
the fetal haplotype was done by relative haplotype
dosage (RHDO). The predicted fetal haplotypes were
in agreement with known outcomes in each of the
nine and 16 pregnancies tested. Importantly, by
combining these two assays into a panel [and with
assays for cystic fibrosis (CF) and congenital adrenal
hyperplasia (CAH) in development], the authors
state that it is possible to test up to three patients
simultaneously for any of three single gene dis-
orders. This significantly reduces the cost to the
point that the DMD/BMD and SMA tests are now
available as a clinical service.
The RHDO method has also been adopted in a
recent study for NIPD of CAH [10], with the fetal
genotype correctly predicted in all five cases
reported so far. Haplotype-based NIPD of CAH has
been explored previously with similarly promising
results, as summarized in a recent review article by
Kazmi et al. [11].
Haplotype-based methods have been used in a
slightly different way by Zeevi et al. [12]. They
demonstrated a universal strategy for NIPD in the
Ashkenazi-Jewish population using SNPs to con-
struct a haplotype around a prevalent mutation
on a common haplotype background in that
particular population.
DROPLET DIGITAL PCR
There have been several studies that have utilized
droplet digital PCR (ddPCR) for NIPD. ddPCR is
based on partitioning of the sample into tens to
hundreds of thousands of droplets, which each con-
tains one or no copies of the target gene. When
compared with real-time PCR, the partitioning
increases sensitivity and specificity because of the
reduced effects of competition of target molecules. A
positive reaction in a droplet indicates the presence
of a single molecule, and the proportion of positive
droplets is used to estimate the concentration
of target.
The majority of studies using ddPCR have dem-
onstrated detection of paternal or de-novo disease-
causing mutations including CF (CFTR gene) [13],
neonatal diabetes (KCNJ11) [14] and achondroplasia
&
(FGFR3) [15]. Perlado et al. [16 ] have taken this one
step further and have also looked at detection of
maternal alleles by relative mutation dosage
(RMD). One difficulty with validating NIPD assays
is the availability of clinical samples for rare disorders
and individual mutations, which they have
addressed by carrying out a proof of principle study
using a SNP strategy to mimic the inheritance pattern
76 www.co-obgyn.com
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of monogenic disorders. They performed NIPD SNP
genotyping in 55 maternal plasma samples, achiev-
ing 100% accuracy for detection of paternal alleles
and 96% accuracy for diagnosis of alleles with
maternal origin by RMD. They conclude that
their study demonstrates the potential of ddPCR
for NIPD fetal genotyping independently of inher-
itance pattern.
BESPOKE/TARGETED NEXT-GENERATION
SEQUENCING
Two recent publications describe the use of targeted
next-generation sequencing (NGS) for NIPD of
&
skeletal dysplasias [17,18 ]. Skeletal dysplasias may
be diagnosed by ultrasonography, and therefore a
noninvasive test is beneficial for diagnosis without
putting the pregnancy at risk. NIPD can be a useful
aid to clinical management as it allows a definitive
diagnosis, differentiation between lethal and non-
lethal forms, and the option of a surgical termin-
ation as a postmortem is not required. It can also be
helpful in twin pregnancies to avoid putting the
unaffected twin at risk. Alternatively, there may
be a family history of the disorder, and where this
is apparently de novo (i.e. not detected in either
parent), or paternally inherited, then NIPD for the
mutation can be offered very early in pregnancy.
Chitty et al. report on the development of an NGS
panel covering 29 known disease-causing mutations
in the FGFR3 gene, associated with achondroplasia
and thanataphoric dysplasia. Dan et al. developed a
targeted capture sequencing method for de-novo
mutation detection in 16 lethal skeletal dysplasia
genes. They analyzed maternal and paternal
genomic DNA alongside maternal plasma cfDNA
for each pregnancy, using a bioinformatics algor-
ithm developed for low-frequency mutation detec-
tion and a strict variant interpretation strategy to
identify de-novo mutations in the plasma data.
Their assay was shown to be successful in three
pregnancies in which variants identified were
shown to be identical to those identified in amniotic
fluid from the pregnancy.
OTHER METHODS
Galbiati et al. [19] looked at the application of
COLD-PCR and microarray analysis for NIPD for
both b-thalassemia and CF. They tested 87 and 40
prenatal diagnoses, respectively, studying detection
of the paternal allele only. Their results showed
complete concordance in all cases with invasive
prenatal diagnosis. They comment that they would
recommend that NIPD be performed by two inde-
pendent methods and this is why they investigated
Volume 29  Number 2  April 2017
 Noninvasive prenatal diagnosis for single gene disorders Allen et al.
this approach, and that both methods are inexpen-
sive and easy to develop and perform in clinical
laboratories.
To date, very few studies have reported on the
development of NIPD for trinucleotide repeat
expansion disorders. This is largely due to the tech-
nical issues associated with detecting repeat expan-
sions within highly fragmented cfDNA. Direct
detection of paternally inherited normal and inter-
mediate-sized CAG repeats in maternal plasma
has been demonstrated ([20] and references
therein); however, there is difficulty in detecting
large Huntington disease repeats in the expansion
size range.
Several groups have recently published details of
assays developed for NIPD of b-thalassemia. These
reports concerned the use of allele-specific real-time
PCR with and without high-resolution melt analysis
to detect the presence or absence of the paternal
mutation in the maternal plasma [21,22]. These
assays showed a high level of accuracy but again
are only applicable to paternal inheritance and
require different mutation alleles to be present in
the mother and father. Despite the limitations, there
may be benefit in considering these types of assays
in suitable scenarios, due to their relative simplicity
and affordability.
CIRCULATING FETAL CELLS
Although much of the recent work has focused on
cffDNA as a source of material for NIPT, the use of
fetal cells is still an attractive proposition as it offers
pure fetal material (not mixed with maternal) and
nonfragmented DNA, which is more amenable to
whole genome analysis. Previously published
methods for isolating circulating fetal nucleated
red blood cells have included density gradients,
magnetic-activated cell sorting and fluorescence-
activated sorting; however, these techniques have
drawbacks including purity and recovery rate that
mean that they are not suitable for routine prenatal
testing. More recently, there have been several pub-
lications that describe techniques capable of isolat-
ing fetal cells at yields and purities sufficient for
NIPT. These use a two-step enrichment with a
positive enrichment process based on red blood cell
hyperaggregation, followed by negative enrichment
using microfluidic technology [24]. The potential
for these cells to be used for subsequent fetal genetic
analysis was demonstrated by determining fetal sex
through expression of SRY genes.
&
Kanda et al. [25 ] have used a combination of a
modified lectin-based erythroblast isolation tech-
nique and automated image analysis to isolate
pure fetal erythroblasts. Fetal sex was demonstrated
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through amplification of the SRY gene and fluor-
escence in-situ hybridization to determine fetal sex
and to demonstrate trisomy 13, 18 or 21. The
authors estimated that the cost of the clinical pro-
vision of this analysis would be equivalent to cur-
rent NIPT techniques; however, they acknowledge
that it is currently impractical for use as a screening
technique.
TRANSCERVICAL TROPHOBLASTIC CELLS
Bolnick et al. [26] first published their trophoblast
retrieval and isolation from the cervix (TRIC) in
2014, in which they demonstrated that they could
use trophoblast cells accumulating in the endocer-
vical canal at the beginning of pregnancy to accu-
rately determine fetal sex from 5-week gestation. In
contrast with cffDNA, it has been shown that the
yield of fetal cells from TRIC is not affected by
gestational age (5–20 weeks), maternal BMI, parity
or maternal age [27]. The same group has since used
this technique to identify fetal sex in a pregnancy at
risk of CAH [28], determine fetal genotypes using
NGS [29] and to evaluate altered biomarkers to
predict manifestation of perinatal disease [30].
Although this technique does not appear to have
the associated risk of adverse clinical outcomes [28]
that are seen with traditional invasive testing
methods, it could still be considered to be a some-
&
what ‘invasive’ procedure. Pfeifer et al. [31 ] have
developed a technique that extracts trophoblastic
cells from the external parts of the cervix using the
Papanicolaou test technique and were able to dem-
onstrate that these cells could be used as a source of
pure fetal DNA, which could be used for NIPD.
ETHICAL AND SOCIAL CONSIDERATIONS
NIPD using cffDNA in maternal plasma allows safer
and earlier prenatal testing, which is likely to trans-
form prenatal care for couples with pregnancies at
risk of SGDs. Therefore, it is important that the
ethical and social issues of the implementation of
NIPD into clinical practice are fully considered.
Several studies have been conducted using focus
groups and interviews that have included adult
patients [32], support group representatives [33]
and carriers of autosomal recessive diseases, includ-
ing those who have an affected child (living or
deceased) and those who have used NIPD [34–36].
Although opinion is generally positive, with
the main benefits being seen as the low risk and
early provision of testing, several themes of com-
mon concerns were identified. These included
accuracy of results, routinization of testing,
informed consent, increased pressure to have
www.co-obgyn.com 77
Prenatal diagnosis
prenatal testing, increase in terminations and
moral issues related to prenatal testing in general
(need for guidelines for when testing should be
offered; paternal rights) [32– 36].
Much of the NIPT currently available is for
conditions that affect the baby at birth or early in
life. However, given the pace at which the technol-
ogy is moving, it seems likely that testing will be
available in the near future for a much wider range
of conditions, including those with varying pene-
trance and/or adult onset. This raises many more
ethical questions, including which conditions it is
ethically justifiable to test for prenatally and how is
this decided and if we are willing to offer an invasive
test for a condition, should we also offer NIPT?
Deans et al. [37] make the point that if parents are
using noninvasive testing for ‘information only’,
are we invading the privacy of that future child’s
right to make their own decisions about genetic
testing? Many of these questions are addressed
in a study by Bennett et al. [38] looking at the
views of health professionals regarding the use
of NIPD for BRCA1/2 mutations in breast and
ovarian cancer.
COST ANALYSIS AND CLINICAL
IMPLEMENTATION
Cost analyses of NIPD and invasive testing pathways
have been performed [32,39], with one study show-
ing that the total costs of NIPD are dependent upon
the complexity of testing; for straightforward auto-
somal-dominant conditions, NIPD costs less than
invasive testing, whereas for more complicated
NIPD for autosomal recessive and X-linked con-
ditions the total costs of NIPD can exceed those of
invasive testing [39]. It is anticipated that the
reduced risks associated with NIPD will also result
in an increased uptake in prenatal testing. One study
showed that 90% of potential service users would
have NIPD if it were available in comparison with
43.5% who would consider invasive testing [32].
These findings add further complexities to the
ethical considerations surrounding the cost-effec-
tiveness of NIPD for rare conditions, the restriction
of access to safer testing for certain conditions and
the economic implications when the findings are
being used for information only.
CONCLUSION
The current review highlights the recent progress
made with NIPD for monogenic disorders. There
have been many publications in the literature on
a case-by-case basis for many different disorders, and
testing is now gradually being introduced into
78 www.co-obgyn.com
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clinical practice. Studies are ongoing to investigate
the cost and ethical considerations of testing.
It remains to be seen if the development of more
powerful technologies such as single cell sequenc-
ing, a reduction in sequencing costs and evolution
of more robust methods for isolation of single cells
will shift the emphasis and scope of NIPD [40]. In
addition, as testing becomes possible for a wider
range of conditions, many more ethical questions
are likely to be raised.
Acknowledgements
None.
Financial support and sponsorship
None.
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED
READING
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been highlighted as:
& of special interest
&& of outstanding interest
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