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From the Division of Laboratory Genetics, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota
Preimplantation genetic diagnosis (PGD) is a procedure that allows embryos to be tested for genetic disorders before they enter the uterus and before pregnancy has begun. Embryos obtained by in vitro fertilization undergo a biopsy procedure in which one or two cells are removed and tested for a specific disorder. If the cell is unaffected, the embryo from which it was taken is judged to be free of the disorder. The embryo can then be transferred to the uterus to initiate pregnancy. Couples whose children are at increased risk for a specific genetic disorder can benefit from PGD. Some of these couples may have affected family members or family ancestry that puts them at high risk for transmitting a particular disorder to their offspring. PGD is an alternative to prenatal tests such as amniocentesis or chorionic villus sampling and since it is performed before a pregnancy has begun, it may be more acceptable to couples who have either had an affected child, previous termination of pregnancy, or who have objections to termination of pregnancy.
PGD tests have largely focused on two methodologies: fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR). This review will focus on the use of PCR-based methodologies to diagnose single gene disorders in single cells; specifically describing the characteristics and limitations of single cell PCR and mutation detection strategies which have been developed for use in clinical PGD.
The hundreds of cycles of preimplantation diagnosis performed to date
have resulted in the birth of several hundred healthy
children.1
As shown in Table 1
, the genetic conditions for which PGD has been applied are numerous and
the various methods used for diagnosis reflect the heterogeneity of
causative mutations.
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Although FISH has largely superseded PCR for sex determination, the
specific diagnosis of single-gene defects remains dependent on DNA
amplification with PCR. In the case of X-linked disorders, testing of
the specific gene has the added advantage of ensuring that all embryos
free of the mutant gene can be selected for transfer, irrespective of
gender.10, 11, 12
The list of disorders and the particular
mutation detection strategies used for PCR-based clinical PGD
application are given in Table 1
.
Materials and Methods
Essentially there are two laboratory components involved in PGD. The first involves the collection of diagnostic material for testing. This is usually performed in a clinical in vitro fertilization (IVF) laboratory under sterile conditions. A set of micromanipulators linked to an inverted microscope with contrast optics and facilities for extended embryo culture are the minimum essential requirements to carry out diagnostic biopsy procedures. The second step involves the diagnostic test itself, which can be performed in a region of the IVF laboratory, an adjacent laboratory equipped to perform molecular analyses or in a completely separate dedicated molecular genetics laboratory equipped to process single cell samples. Minimum requirements include a PCR preparation area (usually a small, dedicated flow hood), dedicated PGD reagent storage facilities, thermal cycler, and access to the necessary post-PCR mutation detection apparatus. The critical component of the diagnostic step is to minimize the level of contamination and a number of possible laboratory designs and procedures may fulfill this requirement.
Theoretically, diagnostic material can be collected at any
developmental stage between the mature oocyte and blastocyst. To date,
four distinct stages have been targeted; metaphase II oocyte, zygote,
cleavage stage embryo, and blastocyst. The four stages dictate
different diagnostic strategies, each with its own limitations. The
different technical approaches required to obtain the material and the
material itself can affect the success rate of the procedure. The
strengths and limitations of each approach are summarized in Table 2
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At present, polar body biopsy in combination with PCR based assays is performed almost exclusively by one group14, 15 while the majority of PGD centers16 obtain genetic material for PGD by cleavage stage biopsy on the third day following insemination when the embryo has between 6 and 10 cells. At this stage, blastomeres are believed to be totipotent and embryo survival and metabolism appears to be unaffected by biopsy.17 While blastocyst biopsy appears to be a promising approach18, 19, 20 its clinical utility for PGD has yet to be demonstrated in clinical practice.
Diagnostic Methods
The success of PCR in amplifying small quantities of DNA to a level at which they can be visualized and subjected to further genetic analysis has made the technique one of the most important diagnostic techniques in the modern molecular laboratory. Application of PCR protocols to single cell analyses has proved to be challenging but ultimately highly successful, and remains the only means of detecting specific mutant alleles in human preimplantation embryos. The limited amount of template DNA (approximately 7 pg) available in a single diploid cell leads to a number of problems which are rarely, if ever, observed in routine diagnostic PCR (in which a starting amount of DNA template of at least 10 ng is usually available). Problems frequently encountered include an increased incidence of detectable contamination, amplification failure, and extreme preferential amplification of one allele or complete absence of one allele (allele dropout) in heterozygous samples.
Characteristics of Single Cell PCR (SCPCR)
Amplification Efficiency
Amplification efficiencies at the single cell level are generally
lower than those encountered during the routine PCR of DNA samples in
which the amount of starting template may be larger by several orders
of magnitude. Reduced amplification efficiency can be the result of
many problems encountered between sample collection and the PCR
procedure itself. Operator problems such as cell loss during the
delicate process of cell transfer to the tube or spontaneous cell lysis
before the cell entering the tube contribute to amplification failure
or reduced amplification efficiency. Intrinsic factors such as
anucleate or degenerating cells with concomitant absence or degradation
of DNA respectively are more difficult to control. Indeed, blastomeres
from poor quality embryos yield lower amplification efficiencies than
their high quality counterparts21, 22
underlining the
importance of blastomere selection during embryo biopsy. Following
successful transfer of a high quality nucleated cell, the cell lysis
protocol used also influences amplification success. Consecutive rounds
of freezing and thawing in distilled water or boiling do achieve cell
lysis23
but the use of either alkaline or proteinase based
lysis buffers has proved more effective.24, 25, 26, 27, 28, 29
Nevertheless, there is no consensus as to which lysis buffer is the
most effective.16
Contamination
With only one or two DNA molecules present per haploid (second
polar body, oocyte, or sperm) or diploid (blastomere or first polar
body) cell respectively, extraneous DNA can easily lead to a
misdiagnosis in clinical PGD. Contamination is an omnipresent threat in
any molecular diagnostics laboratory but the large number of PCR cycles
required for detectable amplification in combination with a single
genome starting template exacerbates this threat. A series of stringent
experimental practices can be implemented to counter contamination but
there is no guaranteed method of eliminating sporadic contamination.
Sources of contamination are numerous since DNA (particularly in the
form of previously amplified PCR products30
) can exist in
aerosol form and, as such, is likely to be present on all exposed
laboratory surfaces. Such "carry-over" contamination caused by the
inadvertent amplification of PCR products generated in previous
experiments is a cumulative problem and probably the most significant
contamination threat in single cell PCR. To address this problem single
cell reactions should be set up in a room designated for this purpose
(pre-PCR area) and physically separated from the area in which PCR
product analysis occurs (post-PCR area). Pre-PCR areas (including the
cell preparation area, the reagent preparation area, and the PCR set-up
area) kept under constant positive pressure can prevent the entry of
contaminants but much of the cellular and PCR product contamination is
introduced by human traffic. For this reason, dedicated gowns, gloves,
overshoes, caps, and masks should be worn and remain in this room,
together with dedicated equipment such as tubes, racks, and pipettes.
Ideally, a unidirectional work flow prevents the re-introduction of
items from a post-PCR area into a pre-PCR area. Filtration and
autoclaving of reagents, incubation of component reagents of the
reaction mix with restriction enzymes to destroy any PCR product (for
example exonuclease III, Alu I, Hae III, and
Hinf I)31, 32, 33, 34
or the use of a mineral oil
overlay to provide a physical barrier against contamination may be of
some value but the introduction of additional components into any clean
system may be counterproductive and could potentially reintroduce
contaminants. Routine decontamination of work surfaces and equipment
using 10% bleach35
or exposure to ultraviolet light to
destroy DNA is also recommended. Unfortunately, no single strategy can
be considered to be 100% effective or render the continuous monitoring
of contamination levels obsolete. For this reason all PCR reagents,
cell washing, and lysis solutions should be rigorously tested for
contamination before any clinical diagnostic application.
Another measure for contamination control which has been used extensively in infectious disease screening by sensitive PCR but not yet in PGD is post-PCR sterilization. One method uses uracil DNA glycosylase (UDG) to cleave uracil bases from PCR products in which dUTP is substituted for dTTP in the PCR mix. In this way the action of DNA polymerase is blocked exclusively with carry-over contamination products but not native DNA.36 A different technique uses isopsoralen which binds to PCR products such that photoactivation following amplification damages the DNA strand preventing it from functioning as a template in subsequent PCRs.37
Cellular DNA from sperm or maternal cumulus cells (both of which may be
present on the zona pellucida of the human embryo) is another potential
source of contamination but can be largely eliminated by means of
precautions in the IVF laboratory. All cumulus cells must be carefully
removed before biopsy and the embryo checked under an inverted
microscope. Moreover, the use of intracytoplasmic sperm injection
(ICSI) a technique used to introduce a single sperm into the cytoplasm
of the oocyte has circumvented the problems caused by supernumerary
sperm which frequently bind to the zona pellucida in large numbers
following standard insemination techniques.38
The biopsied
blastomeres themselves should be washed through a series of fresh drops
of holding medium known to be contamination-free before transfer to the
PCR tube. The commonly used precautions against contamination are
listed in Table 3
.
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The frequency of allele dropout reported in the literature varies widely and has been reported to be as high as 25% in a clinical PGD case.45 This figure could be considered unacceptably high, but the concept of an acceptable ADO rate is meaningful only when parameters relevant to the PGD case have been assessed. For example, a more accurate ADO rate can be established as more cells are analyzed and a higher ADO rate tolerated with contamination rates close to zero in combination with diagnosis of a homozygous recessive mutation.
Reports suggest that blastomeres generally exhibit a greater ADO rate than polar bodies, lymphocytes, or fibroblasts41, 46 although such differences have not been unanimously reported.47 Observations that amplification rates are generally lower for blastomeres than other cell types even when a nucleus is present48, 49 and the detection of haploidy in an estimated 7 to 15% of blastomeres provide further evidence for a cell-specific effect on the observed frequency of ADO.50, 51
The origins of ADO remain elusive but experimental data supports the causative factors being suboptimal PCR conditions and/or incomplete cell lysis. Adequate denaturation is essential for amplification of both alleles as demonstrated by a reduction in ADO when the denaturation temperature is increased in the first cycles of PCR.27
ADO could also arise from DNA deterioration or damage such as strand breaks caused by endogenous nucleases. As with reduced amplification efficiencies, increased ADO is noted in degenerating cells presumably the result of strand-specific DNA degradation.52 Additionally, access to the target genomic sequence by the primers and Taq polymerase may be restricted by, for example, adjacent G/C rich regions reducing denaturation efficiency or differing degrees of folding perhaps related to the stage of the cell cycle.27 Whatever the exact cause, ADO likely arises in the initial cycles of the primary PCR before the number of target molecules is increased by the process of amplification. Evidence for this suggestion comes from experiments in which different proportions of two separate populations of single cells (each homozygous for a different sized triplet repeat sequence) are mixed, demonstrating that the minority allele is undetectable when the starting template ratio is less than one in four cells.53
Allele dropout observed during conventional nested PCR with ethidium
bromide detection comprises both extreme preferential amplification, in
which the PCR product from one allele is present but at extremely low
levels, and true allele dropout (in which one allele is either absent
or has totally failed to amplify). Enhanced detection methods such as
the use of fluorescent primers40
and SYBR green I
staining54
have shown that a proportion of observed ADO is
due to extreme preferential amplification. A fourfold reduction in ADO
for both lymphoblasts and blastomeres has been reported after switching
from an ethidium based protocol to one using fluorescence
primers.55
However, a significant proportion of true ADO
exists even using fluorescent PCR.33, 42
Such observations
from single cell fluorescent PCR reinforce the need for cut-off values
to distinguish background noise, contamination, extremely low
amplification, preferential amplification, and allele dropout. Examples
of preferential amplification and allele dropout from samples of single
heterozygous cells are shown in Figure 1
.
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Protocols that rely on reverse transcription of abundant mRNA molecules followed by PCR (RT-PCR) and subsequent mutation analysis have been proposed as a means of reducing amplification failures and ADO since multiple targets should not be subject to allele specific amplification failure. Single cell expression assays have been developed for the diagnosis of Marfan56 and Lesch-Nyhan57 syndromes. Such assays could prove valuable for genes that are expressed at the cleavage stage, provided that they are not subject to genomic imprinting and that residual maternally-derived transcripts from the oocyte or alternatively spliced products57 do not confuse the diagnosis.
Several other methods of decreasing ADO include the use of restriction enzymes before PCR to shorten genomic template strands (presumably making them more accessible to the polymerase enzyme during the first few cycles of PCR)58 and the use of Taq/Pwo polymerase mixture (perhaps because of the proof-reading ability of Pwo polymerase).59
In addition to reducing ADO, strategies have been proposed to increase the detection of ADO. One such strategy is the use of linked markers46, 60, 61 which simultaneously controls for contamination.62 Use of one or two linked markers reduces undetected ADO by approximately 50% and 75% respectively and with three linked markers ADO is virtually always detected.46 The use of linked markers carries considerable advantages not only from the point of view of reducing the possibility of misdiagnosis, but also by potentially increasing the number of embryos available for transfer.63 For example, in a homozygous recessive condition, carrier embryos could still be transferred even when the normal allele appears to be absent due to ADO, but a linked marker (or markers) is present. However, the identification and work-up of reliable informative linked markers can be labor intensive and may not always be cost effective for all diseases, particularly when the patient population is very small.
Another strategy used to increase ADO detection is special design of
the PCR assay itself. For disorders in which a triplet repeat expansion
(which is refractory to PCR) is the disease causing DNA sequence
change, an assay based on detection of two normal sized triplet repeat
alleles will prevent transfer of affected embryos when the parental
alleles are informative. In addition, assays in which a single
amplified fragment encompasses both mutations of a compound
heterozygous condition should always allow the detection of allele
dropout in an affected blastomere24, 64, 65, 66
(Figure 2)
. In such cases, ADO of the wildtype allele in carrier embryos will
result in a restriction pattern suggesting homozygosity for one
particular mutation. This result is not possible when the parents carry
different mutations. Such a pattern in a clinical diagnosis would
result in rejection of that particular embryo for transfer since it
would be impossible to distinguish between an unaffected carrier and an
affected compound heterozygous embryo.
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Fluorescent PCR
Fluorescent PCR (FPCR) is fast becoming the method of choice for
laboratories performing single cell PCR for a number of reasons.
Compared with nested PCR, FPCR combines increased sensitivity and
throughput, shorter turnaround time,73
and superior
precision in fragment sizing.62
The use of a laser system
to perform automated fragment analysis with various fluorescent
molecules, each with their own unique wavelength of emitted light,
allows simultaneous discrimination of unrelated, similarly sized
products. Furthermore, the thousandfold increase in
sensitivity74
compared with ethidium bromide allows a
single round of PCR, potentially avoiding the contamination which can
result from multiple tube openings.
The accuracy of automated fragment analysis enables, for example, a
deletion of 3 bp in the 
F508 mutation causing cystic fibrosis, to be
clearly differentiated from the normal allele after fluorescent PCR
(Figure 1)
removing the need for either heteroduplex
analysis75
or lengthy conventional electrophoresis using a
high resolution gel. Several different instruments are available for
such analyses and the technique has been successfully applied to PGD
development and clinical cases in many
laboratories.55, 62, 73
Fluorescent PCR is also compatible
with many established forms of mutation analysis such as
SSCP,76
ARMS,77
and restriction enzyme
digestion.78
Multiplex PCR
By using combinations of unrelated primer sets in one PCR assay
(multiplex PCR) it is possible to amplify multiple loci simultaneously
and attempt to overcome the limitations of the single
cell.62, 79, 80, 81
Providing there is no interaction between
unrelated primers or PCR products, the various loci should be amplified
simultaneously within a single reaction. Each multiplex PCR need only
be optimized for the combination of primers involved. Successful
multiplex reactions enable the simultaneous assessment of numerous
loci, with as many as 15 analyzed from larger DNA
samples.82
It may also be possible to assess similar
numbers of loci in single cells but to date the maximum number of
sequences amplified simultaneously from a single cell is seven using
either conventional ethidium detection12
or fluorescent
PCR.62
Unfortunately the problems of allele dropout and
preferential amplification persist even with the FPCR
approach.40, 80
Multiplex PCR can also be used to detect ADO by simultaneous amplification of a disease causing mutation and an informative "linked" polymorphism. This is a particularly useful strategy when diagnosing dominant disorders, but has also been reported for a number of recessive disorders including cystic fibrosis,41 ß-thalassemia,83 and medium chain acyl CoA dehydrogenase deficiency.76 The probability of ADO affecting both mutation site and linked polymorphism is very low and consequently the mutant allele can almost always be detected.
Whole Genome Amplification
One of the most exciting developments in single cell analysis has
been the evolution of protocols designed to amplify the entire genome
from a single cell. Depending on the particular whole genome
amplification (WGA) method used, a starting template of approximately 7
pg of DNA can be amplified up to 1000 times apparently overcoming the
limitation of a single cell.84
The technical difficulties
sometimes associated with multiplex PCR, such as incompatibility of
primer sets and problems distinguishing the various amplified products,
are not encountered using WGA. Moreover WGA provides a supply of sample
DNA that can be reassessed, allowing confirmation of diagnosis using
the same or different methods or the analysis of other genes. The most
commonly used method has been primer extension preamplification (PEP)
which utilizes 15 base oligonucleotide primers of random sequence to
initiate DNA synthesis throughout the genome.85
Reports
estimate that between 70% and 96% of the genome is amplified between
30 and 1000 times.84, 85, 86
PEP has been used to develop PGD protocols for single cell analysis of Tay-Sachs disease,87 cystic fibrosis,88, 89 hemophilia A,90 and Duchenne muscular dystrophy,91 but its clinical application has been limited. One problem is the length of time necessary, since PEP mandates an embryo transfer on day 4 post-fertilization at the earliest; however, a modified protocol has been reported that reduces the time required from >14 hours to 5 hours 30 minutes.92 Nevertheless, PEP was successfully used for PGD in the dominant cancer syndrome familial adenomatous polyposis coli (FAP) allowing the subsequent amplification of two different fragments, one containing a mutation and the other an informative polymorphism.60
Another form of WGA is degenerate oligonucleotide primed PCR (DOP-PCR) which was designed to give general amplification of target DNAs at frequently occurring priming sites, without restrictions due to the complexity of the DNA or the species from which it was derived. It rests on the principle of priming from short sequences specified by the 3' end of the oligonucleotides used, during the initial low temperature cycles of the PCR protocol. Since these short sequences occur frequently and are evenly distributed throughout the genome, amplification of target DNA proceeds at multiple loci simultaneously. Annealing of the specified 3'-most primer sequence is stabilized by the adjacent six bases of degenerate sequence which create a pool of 4096 primers of different sequence, as opposed to the single sequence of a nondegenerate primer. At the 5' end of the primer is a further specified sequence which allows efficient annealing of primers to previously amplified DNA, enabling a higher annealing temperature to be used in later PCR cycles.93 DOP-PCR amplifies a similar proportion of the genome to PEP, but to a much more significant level. A single cell subjected to DOP-PCR can provide enough DNA for over 100 subsequent PCR amplifications.86 Furthermore sufficient DNA is produced to allow additional experimental procedures such as comparative genomic hybridization (CGH) for the detection of chromosome copy number in embryos94, 95 —an approach recently applied in clinical PGD.96
ADO rates after PEP and DOP-PCR are comparable to those obtained by direct amplification of single cell loci.86 A significant drawback of WGA techniques is that amplification of repetitive DNA sequences, such as short tandem repeats, is error prone if performed on WGA products.86, 97 In some studies over 50% of fragments amplified differed from their expected size presumably due to the uniformly low temperatures needed for WGA which could allow slippage of the DNA chain during product generation.86 Such errors would currently rule out the use of WGA for the clinical diagnosis of triplet repeat expansion diseases or diagnoses based on linkage analysis with STRs. The current difficulties associated with the WGA approach will no doubt be overcome because of the enormous potential of the technique to be combined with repeated simplex and multiplex PCR analysis, CGH,86 and microarray analysis.84
Detection Methods
Once DNA from a single cell has been amplified to a detectable
level, most of the mutation detection techniques currently available in
diagnostic laboratories can be used for its analysis (Table 1)
.
Mutation analyses can be broadly divided into three categories: those
that are tailor made for the detection of one specific mutation, those
that detect a variety of different mutations with a single protocol,
and those that do not attempt to detect the mutation but infer the
presence of the mutation. Techniques that fall into the first category
are generally used in a diagnostic context and usually provide a rapid
means of detecting common mutations. Methods in the second category are
known as "scanning" methods and are usually applied to searches for
mutations that have not been characterized. Scanning methodologies are
particularly useful for the diagnosis of inherited disorders caused by
a heterogeneous spectrum of mutations, as a single methodology can
usually be applied for detection of most of the DNA sequence
alterations. The third category, linkage analysis, is frequently used
in the presence of a suitable pedigree, when pathological mutations are
uncharacterized or when known mutations are refractory to PCR. Although
such indications are encountered in couples requesting PGD, the
detection of contamination and allele dropout are becoming powerful
indications in their own right for the inclusion of linked markers.
Amplification Refractory Mutation System
The annealing of allele specific oligonucleotides is the basis of
the amplification refractory mutation system (ARMS) technique. ARMS
employs one oligonucleotide to anneal upstream of the mutation site
while two other oligonucleotides each anneal exclusively to either the
mutant or normal alleles. These allele-specific oligonucleotides merely
serve as primers for PCR and are not detected directly. The presence or
absence of a specific allele is inferred from the presence of PCR
product which is only seen when primer annealing occurs. If nested PCR
is used, the outer set of primers is designed to produce an amplicon
containing the mutation site. Two different inner amplifications are
set up from separate aliquots of the outer reaction, one containing a
primer specific to the normal allele and the other a primer for the
mutant allele. Amplification from only the mutant allele specific
primer would result in the embryo being diagnosed as affected and
excluded from transfer. Heterozygous samples would show positive
amplification for both normal and mutant primer sets. As with
restriction enzyme digestion-based diagnoses, the detection of both
mutant and normal alleles is a safer and more informative test than the
detection of the mutant allele alone. This methodology has been used
for the analysis of the five most prevalent cystic fibrosis mutations
in single cells98
using a nested PCR approach.
A slight modification of this technique which has been applied clinically for the diagnosis of spinal muscular atrophy77 allows different primers specific for mutant or normal alleles to be included in the same PCR mixture. This provides rapid analysis using a single round of PCR. Normal and mutant alleles are distinguishable using automated fragment analysis following fluorescent PCR77 or conventional electrophoresis. The latter detection method requires the design of allele specific primers with different lengths, so called allele-dependent length polymorphism (ADLP), and has been clinically applied for the preimplantation diagnosis of retinitis pigmentosa.99
Restriction Endonuclease Digestion
Amplification of DNA followed by restriction digestion is a common
form of mutation detection in preimplantation diagnosis and has allowed
single cell diagnosis of a wide variety of disorders, generally
involving point mutations.100, 101, 102, 103, 104, 105, 106
With knowledge of the
DNA sequence and the exact mutation, a restriction enzyme may be
selected which will cleave a normal DNA strand while a mutant strand
remains undigested and the products of digestion can distinguished by
electrophoresis. This is the ideal design for a clinical PGD PCR
protocol. Conversely, enzymes which digest the mutant but not the
normal allele can usually be found, but in such cases incomplete or
failed digestion could lead to an embryo being incorrectly diagnosed as
normal.33, 107, 108, 109
For such suboptimal assays the
inclusion of an internal digestion control29
could help to
prevent a misdiagnosis (Figure 3)
.
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Heteroduplex Analysis
Heteroduplex analysis can identify a wide variety of mutations
(particularly small deletions or insertions) and has been used
extensively for identification of the F508 mutation (a 3-bp
deletion) causing cystic fibrosis. Since homozygous samples do not
produce heteroduplexes, F508/F508 affected samples can be
identified through heteroduplex formation following the addition of
equivalent wild-type PCR product and absence of heteroduplex formation
following addition of mutant product.75
As well as
extensive use in PGD of cystic fibrosis22
heteroduplex
analysis has also allowed diagnosis of Tay-Sachs
disease111
and was one of a series of methods used in
parallel for PGD of familial adenomatous polyposis coli.60
One potential problem with this method is the requirement for DNA of
known genotype for mutation detection which could provide an
opportunity for sample mix-up errors.
Single Strand Conformational Polymorphism Analysis
Single strand conformational polymorphism (SSCP) analysis is a
"scanning" assay which is capable of detecting small DNA deletions
and insertions and even single bp substitutions.112
SSCP
has become one of the most frequently used strategies for mutation
detection and, in its simplest form, is uncomplicated and inexpensive
requiring only a minimal amount of equipment. Single strands of DNA,
generated by denaturing a PCR amplified sample, take on sequence
specific conformations that are stabilized by intrastrand interactions.
Allele-specific DNA strands frequently adopt distinct conformations
which migrate at distinct rates when subjected to nondenaturing
polyacrylamide gel electrophoresis. A single protocol can detect a
number of genotypes so long as both mutations lie within the same
amplified fragment. This may simplify the diagnosis of compound
heterozygotes as such samples usually give a unique pattern of bands
easily distinguished from other genotypes. Furthermore, SSCP has been
performed using ethidium bromide (to detect the causative mutation in
PGD of the dominant cancer syndrome familial adenomatous polyposis
coli,60
sensitive silver staining (to diagnose
ß-thalassemia at the single cell level),113
or highly
sensitive fluorescent PCR (to diagnose medium chain acyl CoA
dehydrogenase deficiency).76
A disadvantage of SSCP is
that experimental conditions need to be carefully controlled to ensure
reproducible assay sensitivity. This challenge is exacerbated by the
single cell specific problems such as preferential amplification and
ADO.
Denaturing Gradient Gel Electrophoresis
Denaturing gradient gel electrophoresis (DGGE) is another popular
scanning method which, like SSCP, relies on physical properties of the
DNA strand determined by base sequence. Mutations are detected
indirectly by virtue of altered melting characteristics which affect
the migration of the DNA strand as it passes through a polyacrylamide
gel with increasing concentration of denaturant. The primers usually
used for DNA amplification before DGGE are modified to include a
stretch of approximately 40 guanine or cytosine residues (GC-clamp).
These additional nucleotides significantly increase the proportion of
sequence variants that can be detected in a given DNA fragment.
However, under some circumstances the GC-clamp can reduce the
efficiency of PCR and may actually be refractory to amplification if
used at the single cell level.114
The use of nested PCR
with GC-clamped primers used only in the secondary amplification may
overcome this difficulty. An advantage of DGGE over some other
techniques is its ability to detect multiple mutations within the same
PCR fragment. This has led to its use in clinical PGD for the detection
of mutations causing ß-thalassemia.115
Like SSCP, DGGE
can give banding patterns which are difficult to interpret at the
single cell level.
Despite limitations in applying mutation SSCP or DGGE analysis to single cells, these methodologies can be very useful in identifying mutations in the couple before initiation of a PGD cycle. Other mutation-specific techniques or sequencing then can be used to specifically test for the parental mutations in PGD.
Sequencing
Direct sequencing is accurate, reliable and the time required to
obtain results can be markedly reduced by confining the sequence
analysis (post-PCR) to a smaller region of interest containing the
mutation. Sequencing could be applied as a generic approach for PGD of
any disease involving point mutations, small deletions, or insertions
particularly when a series of mutations lie fairly close together
within the same gene (as is the case for mutations in the ß-globin
gene resulting in thalassemia). Amplification of both parental mutation
sites in the same fragment allows most ADO to be detected and prevent
the transfer of affected compound heterozygous embryos.
Recently, direct sequencing was used in a PGD case involving a novel
skin fragility ectodermal dysplasia syndrome to confirm restriction
enzyme digestion results.66
To attempt PGD, it was
necessary to identify reliably and accurately the presence of the two
different parental mutations (which lead to a functional knockout of
the plakophilin 1 gene) in a single cell assay. Fortunately, the
mutations lay within 11 bp of each other making a nested PCR approach
feasible for the restriction assay, whereby both mutation sites could
be amplified in the same fragment during the first round of PCR.
Restriction analysis was carried out using two separate digests (one
for each mutation). In parallel, cycle sequencing using big dye
terminators on an ABI 310 genetic analyzer (Applied Biosystems, Foster
City, CA) was performed in both forward and reverse primers for
each purified sample (Figure 3)
.
Linkage Analysis
Even when the exact mutation causing a disorder is unknown, the
particular disorder may still be avoided by the detection of linked
markers. Any informative polymorphism, which lies in close proximity to
the disease locus, can be used as a tool to indicate the presence or
absence of the mutation without its direct detection. Markers that are
intragenic or situated close to the gene are preferred for this
approach, as they are unlikely to be separated from the mutation by
recombination during meiosis. To perform linkage analysis, a family
pedigree must be obtained and DNA from family members tested to
determine which polymorphic variant is inherited along with the disease
phenotype. Many types of polymorphism are used for this purpose, the
most commonly used are microsatellites (eg, Simple Tandem Repeats or
STRs). These are highly polymorphic and consequently have the greatest
probability of being informative for a given family.
Prior knowledge of the STR allele sizes of couples undergoing PGD allows the calculation of all possible zygote genotypes. Any deviation from these possibilities indicates the presence of contaminating DNA.62, 80, 116, 117 The polymorphic nature of STR markers also permits the detection of haploidy and uniparental disomy. For these reasons many groups involved in PGD are now attempting to incorporate polymorphic markers into their molecular diagnoses.41, 60, 104 The use of tetranucleotide repeats in preference to dinucleotide repeats and the application of commercially available optimized reaction buffers should reduce the frequency of artifacts known as "stutter bands" that complicate analysis of results.118 The preferred future method for linkage analysis may use Single Nucleotide Polymorphisms (SNPs) which are DNA alterations that occur at a frequency of approximately 1 per 1000 bp throughout the genome. Since variability at a particular locus is limited to the four deoxynucleotides, a large number of SNPs is required for reliable linkage analysis. Microarray analysis (following whole genome amplification of single cells) will be a prerequisite to using SNPs as an alternative to STRs for linkage analysis.
Linkage analysis for PGD has been used for a number of different reasons. The causative mutation may be unknown,119 the sequence containing the mutation may be refractory to PCR,120 or heterogeneity of the causative mutations may make linkage analysis a more cost-effective way to provide a generic test for a disorder.121, 122, 123 In addition, detection of allele dropout (particularly relevant for dominant conditions) and contamination make linkage analysis a powerful tool in clinical PGD. Finally, linkage analysis has allowed non-disclosure testing of embryos for Huntington disease.124 For PGD by linkage analysis, many laboratories rely on informative markers from a two-generation pedigree—frequently available in couples with previous affected pregnancies or children.
The first clinical application of linkage analysis for PGD was to identify the autosomal dominant disorder, Marfan syndrome, in which the specific mutation was unknown. Affected embryos were identified by tracing the inheritance of a dinucleotide repeat polymorphism linked to the causative fibrillin gene.119 Since this application, linkage analysis has also been used to detect embryos carrying mutant alleles of the dystrophin gene125 and has been combined with mutation analysis using multiplex PCR126 or whole genome amplification.60
Diseases caused by the inheritance of large trinucleotide repeat expansions, such as fragile X syndrome and myotonic dystrophy, pose an additional problem for single cell analysis. In these cases the expanded allele is frequently too large to be amplified using PCR or may be subject to in vitro expansion producing erroneous results.53 Consequently, inheritance of the disease allele in a tested blastomere would be inferred by the failure of PCR amplification across the expansion and the absence of the normal allele from the carrying parent. Indeed, conventional electrophoresis and later automated fragment analysis to detect non-expanded alleles was the basis for clinical preimplantation diagnosis of myotonic dystrophy55, 127 and fragile X syndrome.128 Alternatively, linkage analysis may be used with informative markers flanking the expansion. Strategies of this kind have been successfully developed for fragile X syndrome120, 129 and myotonic dystrophy.130 The inclusion of linked markers for the detection of allele dropout has become a standard in some laboratories and such a strategy should have a positive impact on pregnancy rates following PGD since the number of correctly diagnosed embryos available for transfer should increase as ADO is detected.46, 63
Finally, linkage analysis can be of use in exclusion testing as a means by asymptomatic individuals who are at high risk of carrying HD can obtain antenatal genetic testing without incurring the emotional, social, and financial burdens that might result from the presymptomatic disclosure of their own carrier status.124
Methodologies for Future Application to Clinical PGD
Cell Recycling
Another technique, which provides cytogenetic and also molecular
genetic information, is known as cell recycling.131
Fixed
single cells are subjected to sequential PCR and FISH analysis allowing
the investigation of specific gene sequences as well as chromosomal
copy number. This combination of information would be particularly
useful for PGD cases in which patients of advanced maternal age present
with risk for having a child with a single gene disorder. Two clinical
PGD cases have been reported, in which embryos free from the particular
single gene disorder under test, resulted in pregnancies which
miscarried and were found to be trisomy 16132
and trisomy
2266
respectively. In either case, an additional FISH test
to rule out common chromosomal abnormalities would have been
beneficial. Despite its potential, clinical application of cell
recycling is not recommended at present since ADO rates are
significantly higher with fixed template DNA than those observed using
routine single cell protocols.133
Quantitative Fluorescent PCR
Quantitative fluorescent PCR (QF-PCR) assays are based on the
amplification of DNA sequences unique for each chromosome pair and have
been developed to establish the number of specific chromosomes present
in a cell.79
These tests amplify STR or microsatellite
markers with quantitation of products. Although QF-PCR is robust and
reliable and can be completed within one working day, its application
at the single cell level is hampered by an unacceptably high (25%)
rate of preferential amplification which results in artificially skewed
ratios of PCR products and the potential for misdiagnosis of
chromosomal copy number in PGD.80
STR markers can
confidently identify aneuploidy with tri-allelic trisomies in single
cells but this potential has yet to be fully realized owing to a lack
of highly polymorphic chromosomal markers.
Real-Time PCR
Real-time PCR allows the rate of amplicon accumulation to be
measured by detection of fluorescently tagged probes at each cycle of
the reaction. The use of probes directed to either wild-type or mutant
sequence also allows genotyping to be performed. The technique is rapid
and has the added convenience that the amplification and detection
procedures are carried out in the same tube (ie, as a homogeneous
assay), thereby greatly reducing the chances of laboratory
contamination. For example, addition of wild-type or mutant hairpin
probes (which contain a fluorophore and quencher molecule at opposite
ends of the probe) allows accurate mutation analysis as PCR products
accumulate in the reaction tube. As the probes anneal to target
sequence, the fluorophore and quencher are separated and fluorescence
can be measured. The degree of homology between probe and target
determines the particular annealing temperature at which the
fluorescence can be measured. Real-time PCR assays have been used very
effectively to detect multiple copy Y chromosomal
sequences49
(using molecular beacon technology) and
BRCA1 sequences134
(using LightCycler
technology, Roche Diagnostics Corporation, Indianapolis, IN) in single
cells and shows considerable promise for application to clinical PGD.
Denaturing High Performance Liquid Chromatography
This technique provides an efficient and inexpensive method for
the rapid detection of single nucleotide mismatches and small deletions
or insertions within an amplified DNA fragment without the need for
fluorescence. Denaturing high performance liquid chromatography (DHPLC)
exploits the differential retention of double stranded heteroduplex and
homoduplex molecules, allowing the automatic comparison of PCR
amplicons for variation.135
A recent study136
analyzing the CAG repeat region of the Huntington gene in single
fibroblasts and blastomeres using this technology showed promising
results in terms of amplification efficiency and ADO rates. However,
aside from the markedly lower cost when compared with fluorescent PCR
technology, it is difficult to see the advantages this technique can
provide as fluorescent PCR becomes more readily available for routine
molecular diagnostics in laboratories.
Microarrays
DNA microarrays (chips) are one of the latest and most promising
tools for genetic analysis. These chips offer the possibility of
simultaneously analyzing thousands of predefined DNA sequences and can
be applied to DNA diagnostics, gene expression analysis, and aneuploidy
detection. The most significant application has been in monitoring
expression profiles to deduce genes relevant to particular disease
pathologies (by comparing cDNA extracts from tissues derived from
normal or disease states). Detection of aneuploidy using chip
technology would work in a similar fashion to that of expression
analysis.137
Pieces of genomic DNA from specific
chromosomes act as probes on the slide and a competitive hybridization
process between samples from known normal karyotype and unknown occurs.
Aneuploidy detection using microarrays is proving to be more difficult
than expression analysis because copy number changes seen in
aneuploidies are more subtle than gene expression changes which can
vary by orders of magnitude.138
Several methodologies for mutation analysis using microarrays have also been described. One of the more common examples of this is minisequencing in which an oligonucleotide is attached to the chip by its 5' end. Each spot on the surface of the chip can contain several million of these oligonucleotides. The oligonucleotide is complimentary to the sequence of a disease causing gene and its 3' end terminates at the base before a known mutation site. When the surface of the chip is exposed to sample DNA with DNA polymerase and di-deoxynucleotides triphosphates, the sample DNA acts as the template for the extension. By labeling each ddNTP with a different color it is possible to determine which nucleotide was added indicating the presence or absence of the mutation. Solid-phase minisequencing following whole genome amplification by PEP correctly genotyped single cells at 96% of the nucleotide positions analzyed.84 Current drawbacks to using microarray technology in PGD include high cost, poor reproducibility, complex and lengthy data analysis, and the absolute requirement for some form of whole genome amplification.
Quality Control and Quality Assurance for Single Cell PCR
Reliability and accuracy in any molecular genetics diagnostics
laboratory rely on stringent quality control (QC) and quality assurance
(QA) measures, many of which are specific to PCR.139
Such
routine QC and QA measures would include appropriate assay validation,
participation in proficiency testing surveys, testing of reagents
before a clinical case, incorporation of measures to prevent and detect
sample mix-up or contamination, routine equipment maintenance, and
laboratory accreditation. The costs of these standard measures are
generally absorbed within a general quality assurance plan in larger
reference diagnostic laboratories. To ensure the highest standards of
analytic reliability and accuracy for single cell analyses, additional
measures are required (Table 5)
. The combination of general and single cell specific QC/QA costs could
be prohibitive in IVF laboratories providing single cell diagnostics.
|
In view of the cost of PGD treatment and the unique nature and origin of the test material, the additional cost and inconvenience to the patient of pre-cycle screening to ensure appropriateness of testing is justified. Before commencing a PGD cycle, it is vital to verify the DNA diagnosis using peripheral blood from the couple. Furthermore, it is prudent to apply the specific PGD test to DNA or single cells from the particular couple to discover any unexpected test results which could render future blastomere results questionable (for example, a polymorphism which may exist under a primer used in the single cell assay but not in the routine laboratory assay).
A number of QC processes apply to the single cell PCR procedure itself
relating to amplification failure, contamination (Table 3)
, and allele
dropout (Table 4)
. With respect to amplification failure, intrinsic
problems relating to the biopsied material can be reduced by selecting
only mononucleate blastomeres for analysis and using a check gel (when
appropriate) to avoid time-consuming and costly post-PCR processing of
failed samples. This latter measure is particularly important in view
of the high number of samples expected to have no amplification (ie,
wash blank controls). The number of blanks to include in assay
development and clinical cases presents something of a dilemma in view
of the calculation that 300 negative blanks are required to ensure that
the contamination rate is less than 1%. A two-stage testing procedure
has been suggested to maintain this low contamination rate. Before
clinical implementation, a large series of blanks (eg, 100) should be
run. After this, smaller series should be run
periodically.63
Specific QC and QA measures taken for
mutation detection procedures have been discussed in previous sections
of this review and are listed in Table 5
.
Finally, the documentation and hand-over procedures in clinical PGD must be stringent to avoid sample mix-up at any stage of the process. Such procedures are critical for the reporting of any genetic test result. Fortunately, some of the more recent technologies (such as real-time PCR) should make it possible to avoid transfer of material between tubes since amplification and mutation detection takes place in the same tube. Regardless of this possibility, it is recommended that critical procedures (eg, transfer of embryos between dishes) be witnessed by a second person and a rigorous protocol for labeling tubes and loading gels be implemented.
One QA measure, conspicuous by its absence in clinical PGD at present,
is external quality assessment (EQA) or proficiency testing. If
satellite PGD (in which IVF laboratories collect embryonic material for
analysis at distant diagnostic laboratories) is to become more
accepted, EQA is essential to maintain the highest standards of patient
care. The different mutation detection strategies used to diagnose the
same disorders (as shown in Table 1
) demonstrate the lack of consensus
and standardization in PGD. An analogous area of genetic testing is PCR
screening for Y chromosomal microdeletions in the work-up for male
infertility in which an EQA project is providing laboratories worldwide
with overall misdiagnosis rates and an individual performance
rating.140
Organizing a similar scheme for PGD is
essential but represents an enormous challenge which may ultimately
only be met under the auspices of such organizations as the ESHRE PGD
consortium1, 16
or the International Working Group on
Preimplantation Genetics.141
Ethical, Legal and Social Issues Relating to PGD
Considerable differences in the regulatory oversight of PGD exists among countries, ranging from total bans on any embryo manipulation to the almost complete absence of any regulations or authority.142 The high cost of practice, low pregnancy rate,143 problems with patient access, and insurance coverage appear to be the biggest drawbacks to universal acceptance in societal terms. Ethical discussions considering the moral status of the human embryo144 and what constitutes severe genetic disease have been debated elsewhere145, 146 but such discussions are clearly outside of the purview of this methodological review. Somewhat reassuring for those centers currently offering PGD is the acknowledgment from professional organizations that PGD can now be considered a "standard of care" rather than an experimental treatment.147
Conclusions
Robust and reliable single cell PCR diagnoses require optimization of reaction conditions and appropriate mutation detection strategies. For this to be achieved, one must fully appreciate the difficulties of amplifying DNA from a single cell. Once the limitations of a single cell have been overcome, using some form of DNA amplification, the mutation detection methods available in the molecular genetics armory are all applicable with only minor modifications.
The use of informative polymorphisms, which can provide confirmatory results to mutation analysis, identify contamination and increase the detection rate of ADO, will increase the reliability and accuracy of many of the diagnostic strategies already reported. For single cell applications, fluorescent PCR will likely replace conventional PCR strategies in view of its speed, throughput, and sensitivity, all of which could help to reduce the cost of each diagnostic test. Increasing amounts of chromosomal information from single cells will also be obtained using PCR-based techniques such as improved methods of quantitative fluorescent PCR and whole genome amplification in combination with comparative genomic hybridization or microarray technology. Whatever the difficulties faced by single cell diagnosis, the growing patient demand for PGD will continue to drive research into the application of further strategies for the diagnosis of an increasing variety of inherited diseases.
As can be seen from Table 1
, a large number of assays have been
developed over the past decade to detect a variety of disorders.
Development of any single cell assay can be costly and time-consuming
and the development of assays for couples with unique mutations is a
tribute to the dedication of researchers in the field of
preimplantation genetics. However, the focus of the next decade should
be to develop robust single cell assays with an emphasis on making such
tests generic (for example, using linkage analysis with STRs or SNPs)
to use limited resources cost-effectively to help more couples. Each of
the various mutation detection methods and PCR strategies described
above is associated with a different turn-around time (for example,
real-time PCR requires less time than nested PCR followed by
restriction digestion). The improvement of embryo culture medium
supporting embryo development to the blastocyst stage now provides up
to 3 days for analysis, more than enough time for any of the methods
described above. Expansion of the analytic window has also made
possible the geographical separation of IVF center and diagnostic
laboratory, although significant logistics issues remain.
In conclusion, PGD testing is largely unregulated by any accrediting agency at present. The introduction of standardization, proficiency testing, and external quality assessment procedures among centers offering PGD (whether on-site or at a satellite laboratory) is in accordance with other forms of molecular testing and would ensure the highest quality of care for all patients.
Footnotes
Address reprint requests to Dr. Alan R. Thornhill, Mayo Clinic, Department of Laboratory Medicine and Pathology, Division of Laboratory Genetics, 200 First Street SW, Rochester, MN 55905. E-mail: thornhill.alan{at}mayo.edu
Accepted for publication November 28, 2001.
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X174 DNA/Hae III marker,
Promega Corporation). Lanes 1–10:
Single lymphocytes heterozygous for R650X mutation in LAMA3
gene. Au and Bu, uncut
LAMA3 and LAMB3 PCR products, respectively.
Allele dropout (*) is
apparent in lanes 2, 7 and 8. The
internal digestion control prevents an affected cell being misdiagnosed
as unaffected (as a consequence of failed
Dde I digestion) since the
LAMB3 product remains undigested
(Bu)
at 461 bp.