HMB265H1 Summer 2016 Assignment

HMB265H1 Summer 2016 Assignment

Assignment Objectives:
· apply genetic concepts to a current disease/condition
· develop an appreciation of human genetic diseases/conditions
· summarize and interpret scientific data
· create a family tree/pedigree
Part A: Refer to the list of sources below and write a 500-word, 1-page essay about the genetics
of one of the single-gene diseases/conditions mentioned in the article by Lench, N. et al. “The
clinical implementation of non-invasive prenatal diagnosis for single-gene disorders: challenges
and progress made.” You are expected to synthesize information from all sources listed below
and write in your own words. Chunking text with minimal changes (i.e. copy-and-pasting
phrases) is not acceptable; see below on “Academic Honesty”.
Part B: Create a possible family tree for the disease/condition you have chosen to write about in
Part A. The family tree should contain four generations (more is OK, please limit to 6): include
at least 12 children in one of the generations, and at least 2 affected individuals in the pedigree.
Include a figure title and a figure legend/caption that contains a description of the figure in 2-3
sentences. Use the pedigree symbols provided in the lecture notes and textbook. You can also
use the symbols provided below:
unaffected affected unaffected fetus unaffected fetus affected fetus affected fetus
fetus fetus with stillbirth/ terminated with stillbirth/ terminated
miscarriage miscarriage
Due Date: Part A of the assignment must be submitted electronically to the Turnitin web site by
Thursday, June 09th before 6 pm (use the Turnitin tab on the Blackboard website menu; read
“Academic Integrity” at this tab before submitting). An identical electronic version of Part A
must also be uploaded onto Blackboard. As well, you must hand in an identical paper copy of
Part A, along with Part B, to your TA at the beginning of your tutorial on June 9th. Please
include your Turnitin receipt number on the front page. Failure to submit either version on
the assigned day and time will result in a mark of zero for the assignment.
This assignment is worth 10% of your final course grade. Please follow the instructions
carefully. Not following instructions will result in mark deduction.
Check your assignment with Turnitin yourself before the due date!
To take advantage of this option—submit your assignment to the Turnitin website before the due
date. You can then make changes and submit the revised version by the deadline on June 9th. In
order to guarantee that you can view your results, you should submit at least 2 days in advance.
Remember that even one sentence directly copied from a source (if not in quotation marks) is an
academic offense. See Academic Honesty below.
To submit your assignment (or draft) to Turnitin go to the Turnitin tab on the HMB265
Blackboard course website.
Additional instructions for Part A:
Essay Format: You must use 12-point Times New Roman font, single-spaced text (when
formatted in Microsoft Word), and 2.5 cm (1 inch) margins on all 4 sides. The reference list can
be on a separate page and does not count towards the word limit. A word count variation of up to
10% is allowed. Your essay should have:
ü a title
ü author (your name, student number, TA name)
ü an introduction paragraph
ü body paragraphs (each with a theme with information backing up that theme)
ü a conclusion paragraph.
There should be a logical progression within paragraphs as well as between paragraphs. Please
use your college writing centre for helpful advice.
Sources:
1) Lench, N. et al. The clinical implementation of non-invasive prenatal diagnosis for
single-gene disorders: challenges and progress made. Prenat. Diagn. 33, 555-562
(2013).
2) Two original scientific articles published on the genetics of one of the single-gene
diseases/conditions mentioned in the article (find by going to pubmed at
http://www.ncbi.nlm.nih.gov/pubmed/ and searching using the name of the disease and the
search term “genetics”). These original scientific papers must be primary articles in which
new scientific research is performed and the results are being communicated.
3) One scientific review article published in the last twelve years (from year 2002+) on the
genetics of one of the diseases/conditions mentioned in the article (find by going to pubmed
at http://www.ncbi.nlm.nih.gov/pubmed/ and searching using the name of the disease and
the search term “genetics”).
You can focus on any of the suggested following genetic aspects: Discovery of the gene involved,
mode of transmission (recessive, dominant, other?), how common is the disease in the population,
what the affected gene encodes and what is the normal physiological function of its product,
what is the function (if any) of the product encoded by the mutant allele, why does the variant
increase risk, animal/cell culture models that manipulate the gene to study its effects, gene
therapy or other therapeutic approaches (Note that you should only focus on one or two of these,
not the entire list). The essay should be in the style of a scientific review—not an essay making
an argument.
Start by framing the issue using the article by Lench, N. et al. “The clinical implementation
of non-invasive prenatal diagnosis for single-gene disorders: challenges and progress made.”
(That might be all you need to cite from the article—unless more relevant information is
there). Then give some general information about the disease.
Use the information in the primary papers to focus your essay to a given area. This is where
you can get specific. The majority of your written assignment will consist of material
from these 2 primary papers. There is no need to go into great detail about the methods—
but do mention how the overall experiment was done (e.g. looking at family members
affected with the disease, studies with mice, in vitro expression studies, etc).
Conclude with the big picture (i.e. relate the information from the primary papers to your
introduction of the disease).
References:
1. Use the 4 sources, as described above. (It is OK to use more than these 4 sources, but you
will not receive additional marks for doing so).
2. Cite appropriately. Include a reference list at the end that contains the articles that you
cite in the text. Use the referencing style of the journal Nature for the in-text citations and
for the reference list (see: http://www.nature.com/nature/authors/gta/index.html#a5.4).
3. In scientific writing, quotations (i.e. identical wording enclosed by quotation marks) are
rarely used. At a maximum, we would expect one quotation. All other information must
be written in your own words. The ideas from your sources must be synthesized (i.e.
combined in unique and original ways).
Academic Honesty:
The assignment is to be done on an individual basis, not in collaboration with others. Make sure
you keep your own work in a secure location and do not share with others. In addition to directly
copying from published literature and web sites, copying from others or from your own previous
assignments is considered plagiarism. This type of academic misconduct is representing “as
one’s own any idea or expression of an idea or work of another in any academic examination or
term test or in connection with any other form of academic work”, and is a serious academic
offense (see: http://www.utoronto.ca/govcncl/pap/policies/studentc.html).
Useful guides to help you to appropriately use sources include:
“How not to Plagiarize” by Dr. Margaret Procter at http://www.utoronto.ca/writing/plagsep.html
and “Paraphrasing and Summary” by Jerry Plotnick at
http://www.utoronto.ca/ucwriting/paraphrase.html
To deter plagiarism in written assignments, we are using the service Turnitin. Please see
Blackboard for information on how to submit, and for the course ID and password also.

REVIEW
The clinical implementation of non-invasive prenatal diagnosis for
single-gene disorders: challenges and progress made
Nicholas Lench1
, Angela Barrett1
, Sarah Fielding1
, Fiona McKay1
, Melissa Hill1
, Lucy Jenkins1
, Helen White2 and Lyn S. Chitty3,4,5*
1
NE Thames Regional Genetics Service, Great Ormond Street Hospital for Children, London, UK
2
Wessex Regional Genetics Laboratory, Salisbury District Hospital, Salisbury, UK
3
UCL Institute of Child Health, London, UK
4
University College Hospital NHS Foundation Trust, London, UK
5
Great Ormond Street NHS Foundation Hospital, London, UK
*Correspondence to: Lyn S. Chitty. E-mail: l.chitty@ucl.ac.uk
ABSTRACT
Recently, we have witnessed the rapid translation into clinical practice of non-invasive prenatal testing for the
common aneuploidies, most notably within the United States and China. This represents a lucrative market with
testing being driven by companies developing and offering their services. These tests are currently aimed at women
with high/medium-risk pregnancies identified by serum screening and/or ultrasound scanning. Uptake has been
impressive, albeit limited to the commercial sector. However, non-invasive prenatal diagnosis (NIPD) for single-gene
disorders has attracted less interest, no doubt because this represents a much smaller market opportunity and in the
majority of cases has to be provided on a bespoke, patient or disease-specific basis. The methods and workflows are
labour-intensive and not readily scalable. Nonetheless, there exists a significant need for NIPD of single-gene
disorders, and the continuing advances in technology and data analysis should facilitate the expansion of the NIPD
test repertoire. Here, we review the progress that has been made to date, the different methods and platform
technologies, the technical challenges, and assess how new developments may be applied to extend testing to a wider
range of genetic disorders. © 2013 John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.
Funding sources: Some of the work described in this manuscript presents independent research funded by the National Institute for Health Research (NIHR) under the
Programme Grants for Applied Research programme (the ‘RAPID’ project) (RP-PG-0707-10107) and the Central and East London NIHR Comprehensive Local
Research Network. Professor Lyn S. Chitty is partially funded by the Great Ormond Street Hospital Children’s Charity and the NIHR Biomedical Research Centre at
Great Ormond Street Hospital. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.
Conflicts of interest: None declared
CURRENT STATUS
To date, the main clinical application of non-invasive prenatal
diagnosis (NIPD) for single-gene disorders has been for severe
X-linked conditions affecting male fetuses, for example
Duchenne muscular dystrophy, where it has been shown to
reduce the need for invasive testing by identifying male
bearing pregnancies.1,2 In addition, fetal sex determination
may be used to identify female fetuses in pregnancies at risk
of congenital adrenal hyperplasia, as genital virilisation in
affected female fetuses can be significantly reduced by the
antenatal administration of dexamethasone therapy. On the
basis of detecting the presence or absence of Y-chromosome
sequences in maternal plasma, fetal sex determination does
not present a significant technical challenge. These tests are
widely practised, clinically cost-effective3 and over the last
few years have gradually been implemented into clinical
genetic practice across Europe (Figure 1).4,5
This has been followed by the development of NIPD of
autosomal dominant disorders where the mutation is carried
on the paternal allele, some de novo mutations and recessive
conditions where the parents carry different mutations, and
diagnosis is based on detection or exclusion of the paternal
allele. The principal approach in these examples is generally
polymerase chain reaction (PCR)-based, using a relatively
straightforward procedure to determine the presence or
absence of the mutant allele in the maternal plasma (see
Supplementary Table 1). More recently, attention has focussed
on recessive disorders, particularly the haemoglobinopathies
(sickle-cell anaemia and thalassemia)7,8 and definitive
diagnosis of X-linked conditions such as haemophilia9 where
the mother carries the mutant allele and where there is already
an existing and significant demand for conventional, invasive
prenatal diagnosis. The diagnosis in these situations presents
a number of technical challenges, as there is a high circulating
Prenatal Diagnosis 2013, 33, 555–562 © 2013 John Wiley & Sons, Ltd.
DOI: 10.1002/pd.4124
load of mutant allele from the carrier mother. Nonetheless, a
number of studies based on digital PCR and next-generation
DNA sequencing technologies have now been published that
can discriminate affected from unaffected fetuses7–9 (see
Supplementary Table 1).
CLINICAL UTILITY
In the UK, as in other countries, most prenatal diagnostic tests
are requested because of risk of aneuploidy, and in 2010–2011,
only 2600 of 16 500 were performed because of a risk of a
genetic condition.6 The indication for genetic testing can be
divided into two main groups with the majority being carried
out in pregnancies where the mutation is established, either
because of a known family history or following carrier
screening in early pregnancy. The other group comprise
pregnancies where fetal ultrasound findings suggest an
underlying genetic aetiology. For the latter, molecular testing
is often required to confirm the diagnosis. Until recently,
definitive molecular diagnosis within a timescale, which could
influence pregnancy management, was not often achievable
because any one of a number of different mutations, often in
several different genes, was potentially responsible for the
sonographic findings. For example, although the identification
of short long bones in the third trimester may be indicative of
a skeletal dysplasia, the underlying diagnosis is not always
clear. In this situation, prenatal testing for achondroplasia
has been possible via invasive testing for many years and was
one of the first tests developed using NIPD (see Supplementary
Table 1), as this condition is caused by a single mutation
in FGFR3 in around 98% of cases.44 However, if NIPD is
negative, there remains a range of other conditions with
similar features that cannot easily be excluded, for example
hypochondroplasia, acromesomelic dysplasia, etc, without
further time-consuming and costly testing. There are other
situations involving many more and often very large genes,
for example, the fetus presenting with large echogenic kidneys
where aetiology includes autosomal dominant and recessive
polycystic kidney disease, Beckwith–Wiedemann and
Laurence–Moon–Barbet–Biedl to name but a few.45 In these
situations, knowledge of the underlying diagnosis would be
invaluable in prenatal counselling, but definitive molecular
diagnoses in pregnancy in the absence of a positive family
history remain a challenge.
TECHNICAL CHALLENGES
There are a number of fundamental technical challenges that
need to be addressed before NIPD can be applied more widely
to genetic disorders. These predominantly relate both to the
size of cell-free fetal DNA (cffDNA) fragments, which are
known to be shorter, on average, than maternal cell-free DNA
(cfDNA) fragments,46 and the relative abundance of maternal
cfDNA.47 Interrogation of fetal DNA sequences would be
greatly simplified if there was a reliable, cost-effective method
for selectively enriching and isolating short fragment cffDNA
from maternal plasma. Although some progress has been
made with the development of polymeric microsystems that
combine electrokinetic trapping, isotachophoresis and
capillary electrophoresis,48 these technologies still remain
relatively far-removed from routine implementation within a
diagnostic service laboratory.
Size of cffDNA
The size of circulating DNA has been studied by next-generation
sequencing (NGS), and fragments have been shown to be around
160 bp in size with a small percentage ranging to 340 bp.46 Ychromosome
sequences were generally <150 bp in length,
suggesting that fetal-free DNA is shorter than maternal-free
DNA. This means that there are size constraints for the design
of NIPD assays to detect a given fetal DNA target. When analysing
a single-point mutation, the constraint of the small fetal
fragments can be accommodated easily by designing small
amplicons, for example <150 bp, to ensure efficient amplification
of target mutation. However, for some diseases, it may not be
possible to use NIPD to directly analyse the disease-causing
mutation, as in the case of fragile X syndrome where the disease
can involve large (CGG)n trinucleotide expansions of >1 kb
upstream of the FMR1 gene. NIPD for Huntington disease (HD)
expansions have been reported; however, reliable detection will
be limited inevitably by the size of the polymorphic (CAG)n
trinucleotide repeat in exon 1 of the HTT gene.28,29,31 Triplet
repeats =40 will always give rise to HD, and whereas the lower
expansion sizes (40–60) have potential to be detected, larger
expansions may be undetectable because of the small size of
the cffDNA fragments. In cases such as HD, it may be possible
to overcome this problem by using alternative approaches such
as extensive haplotype analysis (using small amplicons) to map
the inheritance of the affected chromosome.49
Presence and quantification of cffDNA
A critical factor for any non-invasive prenatal test is ensuring
that cffDNA is present in the sample being tested. Currently,
clinical applications of NIPD require the detection of the
paternally inherited (or de novo) allele to make a definitive
diagnosis. Failure to detect the paternal target sequence or de
novo mutation is either indicative of a true-negative result or
could be due to the lack of amplification of the sequence due
to low concentrations of cffDNA or the complete absence of
cffDNA in the sample. Amplification of a fetal-specific marker
that confirms the presence of cffDNA allows a negative result
0
50
100
150
200
250
300
350
400
450
500
2006 –
2007
2007 –
2008
2008 –
2009
2009 –
2010
2010 –
2011
Number of referrals
Figure 1 Trends in the use of cell-free fetal analysis for the
determination of fetal sex in pregnancies at high risk of sex-linked
genetic disorders in the UK
556 N. Lench et al.
Prenatal Diagnosis 2013, 33, 555–562 © 2013 John Wiley & Sons, Ltd.
to be more accurately interpreted as either a true-negative or
false-negative result. The use of several fetal markers has been
reported including Y-chromosome sequences for male
pregnancies, panels of common polymorphic short tandem
repeats,50 single-nucleotide polymorphism (SNPs) or insertion/
deletion markers,51,52 and epigenetic markers such as
hypermethylated RASSF1A promoter.53,54
Y-chromosome sequences (e.g. DYS14 and SRY) can be used
to confirm the presence of cffDNA but only in male
pregnancies. DYS14 is a multicopy sequence present in the
TSPY1 gene and has been used as a target for fetal sex
determination, fetal marker and for quantification of cffDNA.
The multicopy nature of the target does mean that detection
is improved at low fetal DNA concentrations, but some studies
have shown that DYS14 can be amplified at very low levels in
female samples. Therefore, it has been suggested that it should
not be used as a sole marker for fetal sex determination.51,54
The multicopy nature of DYS14 also means that the sequence
is not optimal for fetal DNA quantification.55 SRY has been
used successfully to confirm the presence of fetal DNA and
for fetal sex determination. An alternative approach, also
applicable to female pregnancies, is to analyse panels of SNPs
or insertion/deletions for paternally inherited sequences.
Scheffer et al.
51 were able to confirm the presence of fetal
DNA in 87% of samples tested by using 24 biallelic insertion/
deletion polymorphisms or paternally inherited blood group
antigens. In 2011, Tynan et al.
56 used a universal multiplexed
SNP genotyping method. Restriction digestion of one allele of
92 SNPs allowed the detection of at least four paternal alleles
with 98% sensitivity and 96% specificity. However, this
approach is time-consuming and potentially costly, as it
requires large panels of markers to ensure amplification of at
least one unique paternal allele. Ideally, a universal fetal
marker independent of paternally inherited sequences should
be used. RASSF1A has been shown to be hypermethylated in
the placenta and hypomethylated in maternal blood.53
Restriction enzyme digestion of the hypomethylated maternal
signal allows the fetal-specific hypermethylated RASSF1A
targets to be amplified using simple real-time PCR assays.
RASSF1A has been used successfully as a fetal marker in several
studies.54,57–59
With the development of highly quantitative single-molecule
counting techniques such as digital PCR and NGS, it is now
possible to use these technologies to assess the underrepresentation
or over-representation of fetal alleles (relative
mutation dosage) in cfDNA samples for maternally inherited
mutations and recessive disorders.7 For these tests, it is not
only important to confirm the presence of cffDNA in the
sample but it is also critical to accurately quantify the
percentage of cffDNA present in the sample. Determination
of the under-representation or over-representation of the
mutated fetal allele using a statistical calculation (sequential
probability ratio testing) relies heavily on the accurate
quantification of total cffDNA (fetal load) in the sample. Any
variation in the estimated fetal load has the potential to result
in both false-positive and false-negative results. Techniques
that have been used to quantify the proportion of cffDNA in
samples include methylation-based DNA discrimination using
matrix-assisted laser desorption/ionisation-time of flight mass
spectrometry (MALDI-TOF MS) analysis,60 digital PCR7,8 and
NGS analysis of polymorphic sequences.61 The MALDI-TOF
MS and NGS approaches are more amenable to highthroughput
testing and have been used extensively in noninvasive
prenatal testing for aneuploidy where a cffDNA fraction
of >4% has been suggested as an acceptable proportion of
cffDNA in a sample to issue a reliable result.62 For lowthroughput
testing of single-gene disorders, digital PCR or an
NGS approach that analyses a highly polymorphic locus may
be a more appropriate strategy for determining fetal load.
Sample collection and time to processing
Several factors relating to blood sample collection have been
shown to affect the proportion of cffDNA present in a sample.
Studies using blood collected from pregnant women have
shown that there is an increase in total cfDNA over time (due
to lysis of maternal cells) but that the absolute quantity of
cffDNA remained constant.63 Therefore, the proportion of
cffDNA reduces over time making the time to processing
of samples a significant factor when carrying out NIPD. Some
studies have used blood collection tubes that contain cellstabilising
solutions (e.g. cfDNA BCT tubes (StreckTM)) to
preserve the original proportion of cffDNA when the time to
processing of the samples is greater than 8 h,64,65 whereas
others have shown that the cffDNA fraction is stable for up to
5 days and suitable for use in fetal sexing and RHD assays.66
Where an absolute quantification of cffDNA is needed, the
use of cell-stabilising agents may be beneficial, but for less
complex testing that requires confirmation of the presence or
absence of an allele not present in the mother, provided the
assay includes a control for the presence of fetal DNA,
collection into K3EDTA and timely processing may be
sufficient.
Other issues
Other issues relate to the presence of multiple pregnancies.
NIPD can be used in multiple pregnancies, but unless there
are sonographic signs to indicate which fetus is affected,
invasive testing will be required to determine whether one or
more fetuses are affected in the presence of a positive NIPD
result. Of particular significance when performing NIPD in
early pregnancy is the potential for an ‘empty sac’ to give rise
to erroneous results,2 as the placenta continues to shed fetal
DNA into the maternal circulation after demise of the fetal
pole.67 This emphasises the need for careful ultrasound
scanning before NIPD, not just to confirm the gestational age
but also to look for evidence of multiple pregnancies.
METHODS USED FOR NIPD FOR SINGLE-GENE DISORDERS
A multitude of methods have been utilised for NIPD of genetic
disorders (Supplementary Table 1). For the detection of alleles
that are not present in the mother, the majority are based on
the detection of a single-base substitution, insertion or
deletion and can be either probe-mediated or primermediated,
using PCR to produce a specific amplicon. The
discrimination of an amplicon containing the mutant allele
versus an amplicon containing the wild-type allele can be
Non-invasive prenatal diagnosis of single-gene disorders 557
Prenatal Diagnosis 2013, 33, 555–562 © 2013 John Wiley & Sons, Ltd.
based on fragment length(s), fluorescence detection (via the
incorporation of different florescent labels) or by direct DNA
sequencing of the PCR amplicon (Table 1). Translation into
routine clinical practice for many of these approaches is not
easy; for example, qualitative assays based on agarose gel
electrophoresis lack sensitivity, are prone to contamination
and, in cases where enzyme digest is also used, are reliant on
the restriction efficiency remaining constant, and furthermore,
analysis is subjective with results being recorded ‘by eye’. New
quantitative technologies with a higher sensitivity and
throughput are emerging, bringing NIPD closer to clinical
practice.
Digital PCR
Digital PCR allows for a more sensitive approach to the
detection of low abundance sequences by the discrete
counting of mutant and wild-type alleles present in a sample.
Sensitivity is only limited by the number of molecules that
can be analysed and the false-positive rate of the mutation
detection assay. Digital PCR relies on the ability to perform
each reaction with an average of one template molecule.
Digital PCR can be performed by the spatial separation of
PCR reactions in capillary systems. The first commercial
platform available for digital PCR was the Fluidigm microchip
that utilises nanolitre compartments defined by pneumatic
valves enabling the simultaneous analysis of up to 39 960
reactions per run with the 48.770 Digital Array integrated
fluidic circuit.
The use of this platform has been reported for the NIPD
of thalassaemia,7 sickle-cell anaemia8 and haemophilia9
(Supplementary Table 1), using relative mutation dosage.7 This
approach requires accurate determination of the proportion of
cffDNA in order to calculate the ratio of mutant to wild-type
allele present in maternal plasma and thereby predict genetic
status of the fetus. Whereas this is readily achieved in Xlinked
conditions, such as haemophilia, using Ychromosome
sequences, its application clinically in female
fetuses is limited by the requirement for other of highly
polymorphic markers.8
Droplet digital PCR
The QX100TM droplet digital PCR (ddPCR) system (Bio-Rad,
USA) is able to generate 20 000 individual PCRs in a single
experiment using a conventional TaqManW quantitative PCR
(qPCR) assay. Using a duplex TaqManW assay on this
platform,56 the BRAF V600E mutation was detected down to
0.001% mutant in a wild-type background. The same group
used ddPCR with an assay for the detection of
hypermethylated RASSF1A in cffDNA to quantify fetal load
and have demonstrated the utility of this technology in a
variety of clinical samples with low levels of target sequence.
The RainDropTM ddPCR system (Raindance Technology,
USA) is able to perform digital PCR in millions of picolitre
droplets. Genomic DNA is compartmentalised in droplets at a
concentration of less than one genome equivalent per droplet
together with two TaqManW probes, one specific for the
mutant allele and one specific for the wild-type allele. After
PCR, the ratio of mutant to wild type is determined by
counting the ratio of green to red droplets. This technology is
able to detect one mutant molecule in 250 000 and can
multiplex up to ten tests on the same sample. Pekin et al.
68
demonstrated the utility of the RainDropTM ddPCR system for
the detection of the six most common KRAS mutations,
quantitating a single mutation in a background of 200 000
wild-type molecules using 1 000 000 partitioned reactions.
Digital PCR may offer a more sensitive approach to NIPD,
not just for X-linked and recessive conditions where the
mother carries the mutant allele but also for the detection of
paternally inherited alleles or those arising de novo. In our
UK National Health Service diagnostic laboratory, we have
experience in several conditions including NIPD for
achondroplasia and thanatophoric dysplasia. This is now the
standard of care in pregnancies at risk of these conditions in
the UK. Our initial approach was to use standard PCR-based
approaches,13,38,39,43 but in the presence of low levels of
cffDNA when PCR gave inconclusive results, we have found
digital PCR is more sensitive (Figure 2). This was also clearly
demonstrated in the analysis of a cfDNA from a pregnancy where
the fetus was found to have autosomal recessive polycystic
kidney disease following the sonographic presentation at
36 weeks’ gestation with anhydramnios and very large echogenic
kidneys. In this case, the mother carried a p.Ala1254fs mutation
(c.3761_3762delCCdupG) in PKHD1; the father has not been
found to carry a mutation in this gene, but DNA analysis after
birth showed that the fetus also had a c.9374C>T mutation,
which is predicted in silico to affect protein function, and has
most probably arisen de novo. Analysis of cfDNA extracted from
frozen maternal plasma (taken at 34 weeks’ gestation) using
qPCR for the detection of the de novo c.9374C>T mutation in
PKHD1 gave an inconclusive result, although the detection of
Table 1 Methods used for non-invasive prenatal diagnosis of
single gene disorders
Technique Approach
PCR (end point) Size fractionation of amplicons
PCR (end point) Restriction enzyme digestion
Quantitative fluorescent PCR (end point)
Quantitative real-time PCR Taqman probes; MGB probes
Allele-specific PCR Primer-mediated or probe-mediated
discrimination of alleles
Peptide nucleic acid clamp PCR Primer/primer competitive hybridisation
COLD PCR Differential melt characteristics of
heteroduplexes and homoduplexes
Denaturing high performance liquid
chromatography
Differential melt characteristics of
heteroduplexes and homoduplexes
MALDI-TOF MS Sequence discrimination based on mass
APEX Arrayed primer (single base) extension
Mini-sequencing Fragment sequencing
Digital PCR Single-molecule counting combined
with probe based assays
PCR, polymerase chain reaction.
558 N. Lench et al.
Prenatal Diagnosis 2013, 33, 555–562 © 2013 John Wiley & Sons, Ltd.
a
b
Figure 2 Heat map images showing digital polymerase chain reaction (PCR) run in duplicate, with wild-type alleles labelled red and mutant alleles blue, for
(a) Fraser syndrome samples, with wild-type (WT) signals in all samples but mutant signal (c.10261C>CT in FRAS1) only in the paternal gDNA and cfDNA
for the first pregnancy. No mutant target molecules are present for the second pregnancy (one sample only shown). (b) Autosomal recessive polycystic kidney
disease (ARPKD) samples withWT signal present in all samples but the non-maternal causative mutation (c.9374C>CT on PKHD1) only found in the affected
pregnancy, not in the maternal or paternal gDNA, or the cfDNA from the second (unaffected) pregnancy, indicating that this mutation arose de novo
Non-invasive prenatal diagnosis of single-gene disorders 559
Prenatal Diagnosis 2013, 33, 555–562 © 2013 John Wiley & Sons, Ltd.
the paternally inherited insertion/deletion polymorphism
MID836a confirmed the presence of cffDNA. Subsequently,
digital PCR using the same sets of hydrolysis probes and primers
confirmed that neither parent carried the c.9374C>T mutation
but clearly demonstrated that the fetus was a carrier of
this mutation (Table 2, Figure 1). Analysis of cfDNA taken
at 12 weeks’ gestation in the next pregnancy did not detect
the mutant allele, indicating that the fetus was unaffected
(Table 2, Figure 1).
The potential for increased sensitivity of digital PCR over
PCR is clear as it is challenging to distinguish one mutant
molecule in 1000 using PCR, but for digital PCR, each molecule
is partitioned, and so it should be possible to amplify mutated
sequences at 0.1%. Digital PCR is also thought to be less
sensitive to inhibition; although it is possible that inhibitors
will delay the amplification of a target molecule over a target
threshold in a qPCR reaction, this does not affect the final
count for digital PCR. One of the main disadvantages of digital
PCR using the Fluidigm Biomark is the cost involved in running
a sample. Each 12.765 digital array contains 12 panels; taking
into account the fact that a minimum of two panels are
required for each sample to be tested, two panels for each
maternal, paternal and normal control gDNA, and two no
template control panels, ten panels are required and,
realistically, one chip per test is required. Designing a set of
primers and hydrolysis probes for each family-specific
mutation is time-consuming and costly, but as they are exactly
the same primers and probes that would have been used for
PCR, there is no disadvantage in comparison with this more
traditional method. Whether transferring these analyses to
platforms based on ddPCR technology renders NIPD for these
conditions more efficient and cost-effective requires further
evaluation. However, given the improved sensitivity and ability
to multiplex increased numbers of samples, these platforms do
require further consideration.
Next-generation sequencing
Reliable, reproducible and cost-effective NGS chemistries now
make it feasible to move it out of the research setting into
routine diagnostic laboratories. A number of different benchtop
platforms are now available that are well-suited to a
clinical diagnostic laboratory service, particularly when using
amplicon sequencing approaches to mutation detection, as
they are cost-effective and have short run times. It is likely that
they will provide a more sensitive and flexible approach to
NIPD for single-gene disorders. It is possible to design panels
that can screen for multiple mutations in a single assay, and
samples can be multiplexed to analyse samples from different
patients in a single run.
We have explored the use of NGS for NIPD in a number of
conditions, including skeletal dysplasias due to FGFR3
mutations, Fraser syndrome and the case of autosomal
recessive polycystic kidney disease described earlier, and have
identified a number of advantages of using NGS for the
detection of paternal or de novo mutations over both digital
PCR or qPCR. Firstly, the number of sequence reads obtained
using NGS is much higher, allowing more certainty of a
positive result. Secondly, whereas for each mutation tested
using digital PCR, a new pair of probes specific for the wildtype
and mutant alleles is needed, using NGS, a whole gene
can be sequenced using the same set of overlapping
amplicons. The cost involved in designing a family-specific
primer/probe set for digital PCR is considerably greater than
for NGS. Furthermore, a large number of samples can be
multiplexed using desktop sequencers such as the MiSeq
(Illumina Inc.), reducing the cost per sample for a run. A digital
PCR reaction can be turned around quickly (less than 4 h
including data analysis), and a MiSeq run takes around 8 h;
although both of these technologies are slower and more costly
than PCR, the benefit of increased sensitivity may greatly
outweigh these disadvantages.
CONCLUSIONS
To date, the implementation of NIPD for genetic disorders into
routine clinical practice has been slow. It has been limited by
the rarity of the disorders and the availability of suitable
technical platforms. Whereas the former problem is only likely
to be overcome with time and banking of samples and
collaborative working,70 service laboratories increasingly have
access to the platforms required to deliver NIPD for a wide range
of single-gene disorders, a situation that will be welcomed by
patients and health providers alike.71,72 However, for successful
implementation, it is necessary to look beyond technology
development and address issues such as costs, service regulation
and stakeholder needs. Until now, research specifically
addressing the ethical and psychosocial concerns and issues for
service delivery for NIPD for single-gene disorders has been very
limited, although some work is now emerging.71–75 Concerns
have been raised regarding the potential ease of access and
hence ‘routinisation’ of testing, with health professionals and
Table 2 Estimated target molecules for wild-type and mutant
alleles for the Fraser syndrome and ARPKD cases using digital
PCR and NGS (MiSeq, Illumina Inc)
Digital PCR NGS
Wild-type
counts
Mutant
counts
Wild-type
counts
Mutant
counts
Fraser’s syndrome
Affected pregnancy 120 17 122 176 18 102
Second pregnancy
9 weeks
90 0 139 810 18
Second pregnancy
12 weeks
206 1 41 862 184
ARPCKD
Affected pregnancy 935 86 65 535 10 207
Second pregnancy 361 0 69 048 29
Two samples were tested for the second Fraser syndrome pregnancy because the first
sample was tested early at 9 weeks, the second at 12 weeks. The estimated number
of single copies present in the sample is calculated by the software based on the
number of FAM positive reaction chambers detected, using a Poisson-based
correction of the data.69
ARPKD, autosomal recessive polycystic kidney disease; PCR, polymerase chain reaction;
FAM, 6-carboxyfluorescein.
560 N. Lench et al.
Prenatal Diagnosis 2013, 33, 555–562 © 2013 John Wiley & Sons, Ltd.
service users feeling strongly that NIPD should be offered
through existing specialist services such as genetics units71,72,74
to ensure appropriate pre-test and post-test counselling. The
development of policy and guidelines will be critical to ensure
high quality and equitable service provision. In addition, costs
will have a major impact on how testing for single-gene
disorders is implemented. Hall et al.
76 have raised concerns
that the costs of setting up tests for individual disorders may
limit which conditions are tested for and how many
laboratories are able to offer tests. To promote equity of access
and standardised service delivery, it will be important to seek
formal approval for new tests, explore cost-effectiveness and
encourage further development.
WHAT’S ALREADY KNOWN ABOUT THIS TOPIC?
• Non-invasive prenatal testing of cell-free fetal DNA in maternal
plasma is widely used for aneuploidy detection, fetal RHD typing
and fetal sex determination in pregnancies at high risk of sex-linked
disorders.
WHAT DOES THIS STUDY ADD?
• We describe the current limited use of cell-free fetal DNA analysis
for prenatal diagnosis of single-gene disorders and discuss the
application of new technologies to aid implementation.
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Supplementary Table 1: Non-invasive prenatal diagnosis of single gene disorders currently reported in the literature
Condition Gene Mutation Method Gestation in
weeks
Comment
Achondroplasia FGFR3
p.Gly380Arg
10Restriction digest 30 single case affected
11Size separation & restriction
digest
Single case affected
12MALDI-TOF MS 34 2 cases affected
13PCR-RED 29-35 4 affected, 2 unaffected
14QF-PCR 27 1 affected
Beta-thalassaemia 15Real-time PCR
16
Size separation & PNA-clamp
PCR
ß-globin 17AS-PCR for SNP 11 1 case positive, 1 case negative
ß-globin SNPs 18APEX 10-12 4 carry paternal SNP
3 negative for paternal SNP
ß-globin 19NGS & RHDO analysis 11-12 2 cases, both confirmed as
heterozygous carriers
ß-globin 20Pyrophosphorolysis-activated
polymerization (PAP)
10-18 Paternal SNP allele detected in
maternal plasma in 13 cases
ß-globin 21COLD-PCR 35 cases all correctly genotyped. 22
shown to carry paternal mutation
Haemophilia A and B F8 and F9 9Digital PCR RMD 11-40 7 correctly classified (4 affected, 3
unaffected)
Haemoglobin E ß-globin (ß
E 22Nested PCR & restriction
digestion
8-18 3 cases positive, 2 cases negative
ß-globin

E
ß
41/42 ß
17
23
Semi-nested & nested realtime
PCR
7-23 E
, 15 carrier E ,3 with
41/42 17 17 41/42
,
6 negative
ß-globin (ß
41/42)
7Digital NASS-RMD 18-20 5 correct, 1 incorrect, 4 unclassified
0
-Thalassemia (Hb Barts) a-globin 24Real-time nested qfPCR 8-20 8 carriers, 1 HbH, 2HbBarts, 2 normal
0
-Thalassemia (Hb Barts) a-globin 25Real-time qPCR 12 – 22 61/62 correctly classified as affected
(sensitivity 98.4%). False positive rate
20.8%.
0
-Thalassemia (Hb Barts) a-globin 26Allele specific real-time PCR 8.5-25 33/65 correctly classified unaffected
0
-Thalassemia (Hb Barts) a-globin 27qfPCR 16-41 10/30 paternal allele correctly
excluded
Sickle Cell Anaemia HBB 8Digital real-time PCR 9-24 52 correctly classified, 6 unclassified,
7 incorrect
HBB 20Pyrophosphorolysis-activated
polymerization (PAP)
18 1 case negative for linked paternal
SNP allele.
Huntington’s disease IT-15 (CAG)n
28,29(semi) Qf PCR 13 Negative for paternal allele
30STR 12 Negative for high risk paternal allele
31Fragment & STRs (6) 12 2 show paternal expansion
1 negative for paternal expansion
1 no paternal allele detected
Myotonic dystrophy* DMPK (CTG)n 32Nested PCR 10 1 affected
Congenital adrenal hyperplasia 33
Fluorescent SNPs 11 and 17 1 negative for paternal allele
Cystic fibrosis CFTR 34Restriction digestion 13 Positive for paternal allele
CFTR (p.Arg668Cys,
p.Lys710ter,
p.Tyr1092ter)
35
SNaPshot 12 & 15 Two cases positive for paternal allele
1 case negative for paternal allele
CFTR (p.Phe508del) 36
Single cell STR genotyping 9-11 32 correctly classified (7 positive)
Spinal Muscular Atrophy SMN 36
Single cell STR genotyping 9-11 31 correctly classified (7 positive)
Hb Lepore 37Allele specific Standard PCR 7 1 case, paternal allele detected
Crouzon syndrome FGFR2 c.1040C>G 38Real-time PCR 12 1 negative, recurrence excluded
Torsion dystonia DYT1 (c.904_906del) 39Real-time PCR 7-9 2 cases positive
Apert syndrome FGFR2 c.755C>G 38Restriction digestion 14 & 23 Index case positive, one negative
excluded recurrence
Retinitis pigmentosa (X-linked) RP2 (c.400C>T) 40Sequencing 10 & 19 Positive
Leber congenital amaourosis CRB1 p.Cys896ter 41dHPLC 12 Positive for paternal mutation
Propionic acidaemia PCCB
c.1218del14ins12
42Melt curve & primer extension 12 Positive for paternal mutation
Thanatophoric dysplasia FGFR3 (c.742C>CT
or c.1948A>AG)
43PCR-RED 12-36 3 cases positive. 1 recurrence excluded
at 12 weeks
PCR, polymerase chain reaction; SNP, single-nucleotide polymorphism; NGS, next-generation sequencing; qPCR, quantitative PCR; PNA, peptide nucleic
acid; dHPLC, denaturing high performance liquid chromatography; PCR-RED: PCR-restriction enzyme digest; QF-PCR, Quantitative fluorescent PCR;
AS-PCR, Allele-specific PCR; APEX, Arrayed primer extension; RHDO, Relative haplotype dosage; RMD, Relative mutation dosage; NASS-RMD,nucleic;
acid size selection-relative mutation dosage; STR, short tandem repeat.

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