Download Preimplantation genetic diagnosis today

Diagnosis of genetic disease
in preimplantation embryos
Preimplantation genetic diagnosis today
Alan H.Handyside
Institute of Obstetrics and Gynaecology, Royal Postgraduate Medical
School, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK
Introduction
The use of assisted reproduction techniques allows preimplantation genetic
diagnosis of an inherited disease in early human embryos before fertilization.
Some genetic defects have even been identified before fertilization by analysis
of the first polar body of the oocyte (Verlinsky et al., 1990, 1992). By selective
transfer of only unaffected embryos, couples at risk of having affected children
can avoid the possibility of terminating an affected pregnancy following more
conventional diagnostic approaches later in gestation. Clinical experience is still
limited but worldwide approaching 200 hundred cycles have now been reported
resulting in 50 pregnancies and over 30 births (Table I) (Harper, 1996).
Collecting and biopsying human embryos
Attempts to flush embryos from the uterus following superovulation and conception in vivo have so far failed. In any case, there would always be a risk that a
potentially affected embryo remained in the uterus. Clinical application of
preimplantation genetic diagnosis has therefore exclusively used the methods
of superovulation and in-vitro fertilization (IVF) established for the treatment of
infertile couples. This produces a number of normally fertilized embryos in a
single cycle accessible for genetic screening. Most couples are known to be
fertile since the previous birth of an affected child is often the reason they are
aware of the risk in future pregnancies. Nevertheless, the use of established
ovarian stimulation protocols following down-regulation with gonadotrophinreleasing hormone (GnRH) analogues is appropriate because the probability of
an embryo being affected is high in many cases. Maximizing the number of
embryos screened increases the likelihood of establishing a pregnancy.
Human embryos have been successfully biopsied at cleavage stages on day 3
post-insemination, at the 6-l0-cell stage, and at the blastocyst stage on days 5
or 6 (Dokras et al., 1990, 1991; Muggleton Harris et al., 1995; Pickering and
Human Reproduction Volume 11 Supplement 1 1996 © European Society for Human Reproduction and Embryology
139
A.H.Handyside
Table I. Summary of world clinical experience with preimplantation genetic diagnosis following
in-vitro fertilization (IVF) and cleavage stage biopsy to February, 1995"
Patients
Cycles
Transfers
Pregnancies
Babies
X-linked (PCR)
41
62
53
14
II
X-linked (FISH)
49
70
56
15
II
Single gene defects
Total
59
65
62
21
12
149
197
171
50
34
PCR = polymerase chain raction; FISH = fluorescence in-situ hybridization.
Pregnancy rate = 25% per cycle, 29% per embryo transfer.
Results collated from 14 centres.
"Modified from Harper, (1996).
Muggleton Harris, 1995). Blastocyst biopsy has the advantage that a larger
number of cells can be removed from the outer trophectoderm layer without
affecting the inner cell mass from which the fetus later develops. However, too
few embryos reach this stage and implant after transfer to be clinically viable
for preimplantation genetic diagnosis at the present time. Removal of one or two
cells at the equivalent of the 8-cell stage does not appear to affect development
to the blastocyst stage in vitro (Hardy et aI., 1990) and has proved to be highly
efficient in practice (Ao and Handyside, 1995). The procedure involves dissolving
a hole in the zona pellucida using acidified Tyrode's solution (Figure 1) and then
aspirating the cells with a second larger micropipette. Also, co-culture of the
isolated cells with the biopsied embryo supports limited proliferation and may
provide an alternative to the more difficult procedure of blastocyst biopsy (Geber
et al., 1995). So far there have been no reports of serious abnormalities at birth
following cleavage stage biopsy. Human embryos, like other mammalian embryos,
appear to be able to regulate their development at these early stages presumably
because cells are not yet irreversibly committed to specific fates.
Genetic analysis
The genetic analysis of single cells has been made possible by the development
of DNA amplification methods involving the polymerase chain reaction (PCR)
and fluorescence in-situ hybridization (FISH) for rapid cytogenetic analysis of
interphase nuclei. Single cells biopsied at cleavage stages are therefore prepared
for analysis either by carefully placing the cells in lysis buffer in micro centrifuge
tubes for PCR or by spreading the nucleus on a microscope slide for FISH.
Single cell analysis by peR
To amplify sufficient DNA from a single cell for analysis by conventional gel
electrophoresis, two rounds of amplification are necessary. 'Nesting' the second
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Preimplantation genetic diagnosis
Figure 1. Scanning electron micrograph of a human cleavage stage embryo following zona drilling with
Acid Tyrodes and biopsy to remove one of the blastomeres. Note the apparently double layered appearance
of the zona and the extensive thinning of the outer layer. (Courtesy of Dr George Nikas).
pair of oligonucleotide primers, which define the region to be amplified, internally
to the first pair has several advantages and provides a safeguard for product
contamination carried over into the sample tubes (Figure 2). Theoretically, only
a single molecule could be amplified and give an erroneous result. Following
amplification, many approaches have been used to identify the presence of
different mutations (Table II). Since the aim is to transfer selected embryos on
the same day as the biopsy to maximise pregnancy rates, amplification and
mutation detection have to be completed in 8-12 h.
Pregnancies and births confirmed to be free of the inherited disease have been
established in couples at risk of several common single gene defects including
cystic fibrosis (CF) (Handyside et aI., 1992), Duchenne muscular dystrophy
(DMD) (Liu et aI., 1995) and Tay Sachs disease (Gibbons et aI., 1995).
Preimplantation genetic diagnosis has also been achieved for a specific mutation
causing the rare X-linked condition, Lesch-Nyhan syndrome (E. Hughes, P.P. Ray,
R.M. Winston and A.H. Handyside, unpublished). With CF and Tay Sachs, the
common three base pair (bp) deletion and 4 bp insertion respectively, were
detected by heteroduplex formation (Figure 3). A 3 or 4 bp size difference in
the amplified fragment cannot be reliably discriminated by rapid gel electrophoresis. By mixing the amplified DNA from the single cell with DNA previously
141
A.H.Handyside
01
____ _ __'____Sl.¥.._ _ _ __
-----
------2S----~--
1 -20 cycles 102
11~
~
12
130 cycles 1
Figure 2. Diagrammatic representation of polymerase chain reaction (peR) using nested oligonucleotide
primers. In the first amplification reaction, an outer pair of primers (0 I and 02) annealing to the sense and
antisense strands of the genomic target sequence encompassing the mutation (tIiangles) is pm1ially amplified.
In the second reaction, a second set of 'nested' pIimers (II and 12) annealing to the target sequence internal
to that of the outer primers is used to amplify a smaller fragment from an aliquot of the first amplified
product. Two rounds of amplification is necessary in most cases to amplify sufficient DNA from single cells
for conventional analysis. The use of nested primers reduces non-specific amplification. It is also a safeguard
to prevent carry-over contamination of samples tubes with the final amplification product since the outer
primers should not anneal to this sequence.
Table II. Some methods for detecting mutations in amplified fragments for preimplantation
genetic diagnosis
Method
Typical application
Fragment length differences
Distinguishing gene and intronless pseudogene sequences
Analysis of variable number tandem repeats
Heteroduplex formation
Rapid detection of small insertions or deletions
Restriction digestion
Detection of mutations that eliminate restriction sites
Allele specific oligonucleotides
(ASOs)
Hybridization of normal and mutant ASOs detected by nonradioactive methods
Single strand conformation
polymorphism (SSCP)
Detection of majority of mutations anywhere within the
amplified fragment
Oligonucleotide ligation
Direct detection of point mutations
Microsequencing
Mutation detection by incorporation of radio labelled
nucleotides-avoids gel electrophoresis
amplified from homozygous normal or affected individuals, denaturing the double
stranded DNA fragments and allowing the mixtures to cool slowly, heteroduplexes
are formed in those cases where both normal length fragments and mutated
longer or shorter fragments are present. When these mixtures are electrophoresed,
142
Preimplantation genetic diagnosis
CellI
~
+
NN AA
+
Cell 2
~
+
NN
Cell 3
~
+
+
+
AA
NN
AA
Cell 4
~
+
NN
+
AA
-- -------NN
NA
Amp
failure
Figure 3. Diagrammatic representation of the use of heteroduplex formation for the rapid detection of, in
this example, the common 3 base pair (bp) deletion, L'1F508, causing cystic fibrosis, Amplified product from
each of four single cells is mixed with amplified product from either known homozygous normal (NN) or
affected (L'1L'1) individuals. The pattern of heteroduplex bands obtained with the two mixtures allows all three
possible genotypes as well as amplification failure to be identified on a small polyacrylamide gel run for as
little as 30 min.
the heteroduplexes are significantly retarded on the gel and can be reliably
interpreted for diagnosing the genotype of the cell biopsied from the embryo.
Single cell analysis by FISH
Pioneering studies of the karyotype of early human embryos following IVF
demonstrated a high incidence of chromosomal abnormalities and mosaicism
(Angell et aI., 1986; Plachot et aI., 1987; Jamieson et aI., 1994). The low
incidence of metaphases, with or without the use of spindle inhibitors, and the
difficulty of banding the short chromosomes at these stages, however, make it
impractical to use this approach for diagnostic purposes and prevents analysis of
all nuclei. FISH is equally applicable to both interphase and metaphase nuclei
and the use of multicolour probes allows the detection of more than one probe
simultaneously. Dual FISH with X and Y specific probes have been used clinically
for identifying the sex of embryos in couples at risk of X-linked diseases which
only affect boys (Griffin et al., 1992, 1993, 1994). This approach has now been
further refined using directly labelled probes and an additional autosomal probe
to distinguish sex chromosome aneuploidy from abnormal ploidy. Combined
with a new spreading procedure involving dissolving the cytoplasm of the single
cells and attaching the nuclei to poly-L-lysine-coated slides, it is now possible
to complete the analysis within 2 h (Harper et aI., 1994).
In addition to identifying sex in X-linked disease, FISH analysis is being used
for detecting trisomies in couples in which one partner is carrying a reciprocal
or Robertsonian translocation. These couples are often at high risk of trisomic
pregnancies and many have repeated miscarriages. These couples are good
candidates for preimplantation genetic diagnosis since several embryos can be
screened in a single cycle. The usual probes specific for repeated sequences in
143
A.H.Handyside
the centromeric region of particular chromosomes may not be suitable and more
distal unique sequence yeast artificial chromosome (YAC) or cosmid contig
probes are being developed. Combinations of probes, specific for the same
chromosome, can also be used for accurate detection of particular trisomies, for
example, in women believed to be gonadal mosaics (c. Conn, J. Harper and
J.D.A. Delhanty, personal communication).
Analysis of spare human embryos or those rejected for transfer following
FISH analysis of the sex chromosomes has confirmed the relatively high incidence
of aneuploidy (-25%) at conception (Munne et al., 1993). There also appears to
be an unexpectedly marked increase with maternal age. In some studies this
increased to almost 50% above the age of 40 (Munne et al., 1995). As the
majority of aneuploidies arise during maternal meiosis especially meiosis I, first
and/or second polar body analysis with combinations of probes detecting X, Y,
21, 18 and 13 are being used to screen embryos in older women undergoing IVF
(Verlinsky et al., 1995). If sufficient euploid embryos can be identified this may
improve pregnancy rates and decrease miscarriage rates in these women.
Analysis of all nuclei in individual embryos at cleavage stages has confirmed
that many embryos are chromosomally mosaic. In addition to aneuploidies arising
during gametogenesis and abnormalities arising at fertilization, it is clear that
there is also a high incidence of postzygotic abnormalities (Delhanty et al.,
1996). Most of the nuclei involved, however, have an abnormal ploidy. For
example, tetraploid nuclei are relatively common although haploid and triploid
nuclei have also been detected. The origin of these cells is not known but it is
possible that in some cases it results from delayed fertilization or pronucleus
formation by supernumerary spermatozoa for example. Non-disjunction in early
cleavage has been observed but is relatively uncommon. Finally, some embryos
have apparently chaotic chromosomal complements in almost every nucleus and
this appears to be associated with particular patients as it is consistently observed
in successive cycles of IVE Human embryos like those of lower vertebrates and
invertebrates, therefore, may lack the normal cell cycle checkpoints and accumulate chromosomal errors during cleavage (Delhanty and Handy-side, 1995).
The accuracy of preimplantation genetic diagnosis
The accuracy of preimplantation genetic diagnosis remains to be assessed in
clinical practice. Three misdiagnoses have already been reported (Harper and
Handyside, 1994). However, these involved different diagnostic procedures and
probably occurred for different reasons. All five children resulting from a recent
series of 18 preimplantation genetic diagnosis cycles for the predominant ~F508
deletion causing CF were confirmed to be homozygous normal (Ao et al., 1995).
However, analysis of each blastomere from the embryos which were not
transferred did reveal some errors particularly in amplifying both parental alleles
in heterozygous carrier cells. Often one parental allele failed to amplify, apparently
randomly (allele dropout), resulting in an incorrect diagnosis as homozygous
144
Preimplantation genetic diagnosis
normal or affected. Further work with single heterozygous lymphocytes has now
shown that this phenomenon is partly explained by incomplete denaturation of
the genomic template DNA during the initial cycles of PCR (Ray and Handyside,
1996). Raising the temperature in the initial cycles improves the efficiency of
denaturation and minimizes but does not eliminate allelic dropout (Figure 4).
Fortunately in an autosomal recessive condition, allele dropout cannot cause
a serious misdiagnosis leading to the transfer of a homozygous affected embryo
(at least in these couples in which both partners were carrying the same mutation).
For compound heterozygote detection or dominant conditions, however, this
could theoretically occur in half the cases of allele dropout in which the affected
allele did not amplify. One way of avoiding these errors for diagnosis of
compound heterozygotes is to amplify a single DNA fragment encompassing
both mutations. This has recently been demonstrated for ~-thalassaemia: major
diagnosis with single lymphocytes from a boy who is a compound heterozygote
for two common Indian mutations of the ~-globin gene (Ray et ai., 1996). A 208
bp fragment including ex on 1 and part of intervening sequence 1 encompassing 12
of the common ~-thalassaemia mutations and also the mutation causing sickle
cell disease was amplified with high efficiency from single cells (Figure 5).
Allele dropout did occur in a minority of cells but in these cases the genotype
appeared to be homozygous at one locus for the normal allele and at the other
for the mutation. Since this is an impossible combination of parental alleles the
possibility of an error is avoided (Figure 6). Clinical application of preimplantation
genetic diagnosis for couples at risk of ~-thalassaemia, many of whom carry
different mutations, should therefore be possible using this approach.
Four general sources of errors have been identified with single blastomere
analysis at cleavage stages (Table III). However, careful monitoring of FISH or
amplification efficiency and contamination levels and reducing allelic dropout
I <Xl
0
80
Q
..rt
<
~
~
60
40
20
0 87
90
(n=50)
93
(n=62)
96
(n=78)
Denaturation temperature
Figure 4. Graph demonstrating the relationship of the incidence of total amplification failure and randum
allele dropout (ADO) with varying denaturing temperatures in the first cycles of nested polymerase chain
reaction (PCR) for a series of single lymphocytes heterozygous for the Ll.F5<Hi deletion. Amplification
efficiency. i.e. amplification of at least one allele. is high at all temperatures (top line) whereas the incidence
of ADO is significantly reduced by raising the annealing temperature to 96°C (bottom line). This suggests
that ADO is caused by a combination of failure to denature the genomic target and genomic target
degradation in the first cycles of PCR.
145
A.H.Handyside
Table III. Sources of errors in preimplantation genetic diagnosis
Potential consequences
Source of errors
PCR failures
Amplification failure
No diagnosis. Misdiagnosis of deletions
Allele dropout (ADO)
Misdiagnosis of carriers.
Preferential amplification
Misdiagnosis of carriers
Non-specific amplification
General misdiagnosis
Carryover contamination
General misdiagnosis
Extraneous contamination
General misdiagnosis
FISH failures
Hybridization failure or
extraneous signals
Misdiagnosis of sex with single probe. Misdiagnosis as
aneuploid
Nuclear abnormalities
Anucleate blastomeres
No diagnosis. Misdiagnosis of deletions
Binucleate blastomeres
Arise by failure of cytokinesis. Not known to cause errors
Multinucleate blastomeres
Mostly fragmenting nuclei. Misdiagnosis likely
Apoptotic or degenerating
nuclei
General misdiagnosis
Chromosomal abnormalities
Haploid
Misdiagnosis of carriers
Triploid
Depending on origin probably not a risk
Tetraploid
Depending on origin probably not a risk
Aneuploid
Only a risk if involving the chromosome with the defect
Chaotic
High risk of misdiagnosis
......0 - - - - - - - - - - - - - -
772bp - - - - - - - - - - - _ . ,
""
...
o O - - - - 2 0 8 b p - - - - I.~"
.
~
5'
~
-:-~:
I
If'
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nt 1:JCnI
Exon 1
J
5:
IVS 1
I
Exon~--,--.:..:..::..=IVS'2-;/-
I
pxon3
1
3'
Figure 5. Diagrammatic representation of the position of the nested primers (outer primers: 01 and 02;
inner primers: [3 and 14) used to amplify the first exon and part of intervening sequence [ (IVS-I) of the
0-globin gene from single cells for the preimplantation diagnosis of 0-thalassaemia. The smaller inner
amplification product of 208 bp encompasses the sites of 12 common mutations including two Indian
mutations at IVS-I nucleotide 1 and 5 (nt 1 and nt5). and the mutation causing sickle cell disease (0').
by manipulating amplification conditions together with analysis of two cells from
each embryo where possible should minimize the risk. Estimates of the risk
based on analysis of several hundred single blastomeres from the series of
preimplantation genetic diagnosis cycles for the common LlF508 mutation causing
CF (see above) ranged betweeri 0.1-2% depending on the genotype and whether
one or two cells are analysed (PRay and A.Handyside, unpublished). At the
146
Preimplantation genetic diagnosis
PARENTS
POSSIBLE
OFFSPRING
APPARENT
GENOTYPE OF
COMPOUND
HETEROZYGOTE
'-y-J
---+ nonsense
ADO
Figure 6. Diagram illustrating the consequences of allelic dropout (ADO) when attempting to identify
compound heterozygotes. Following amplification of a fragment encompassing both mutations from a single
compound heterozygous cell, ADO results in failure to amplify an allele with one of the parental mutations.
However. as the cell cannot be simultaneously homozygous for one mutation and homozygous for the
normal allele at the other site, this result prevents any risk of the embryo being considered for transfer.
lower end of this range this is probably acceptable though it should be possible
to improve on these results and reduce the risk to closer to one in a thousand.
In view of the regulative capacity of embryos at these early stages and the
possibility that the fetus will be derived from only a subset of cells, however, it
may never be possible to exclude errors. For most couples, considerably reducing
their risk of an affected pregnancy is sufficient especially if the a priori risk is high.
The future of preimplantation genetic diagnosis
The development of techniques for genetic analysis of single cells has given us
powerful tools to study early human embryos and understand the contribution of
chromosomal and other genetic abnormalities to developmental failure at these
early stages. Polar body screening and screening of blastomeres from cleavage
stage embryos offer the prospect of improving selection of viable embryos for
transfer which should increase implantation and pregnancy rates in infertile
couples following IVF.
For preimplantation genetic diagnosis in couples at risk of inherited disease,
initial clinical experience and recent developments in the technology of single
cell analysis, offer the prospect of accurate diagnosis of a broadening range of
conditions. These developments include the use of fluorescent analysis of peR
products allowing multiplex analysis of several fragments amplified in the same
147
A.H.Handyside
Table IV. Genetic disorders prevalent in the UK"
Recessive
Haemochromatosis
1 in 500
Cystic fibrosis
1 in 2000
Sickle cell anaemia
1 in 400
Thalassaemia
1 in 400
Pheny lketonuria
I in 10000
Dominant
Huntington's disease
I in 10 000
X-linked
Duchenne muscular dystrophy
I in 4000 males
Complex multifactorial
Cardiovascular disease
> 10% lifetime risk
Alzheimer's disease
10% over 65 years
Breast cancer
10% familial
Colorectal cancer
1 in 200 (HNPCC)
I in 8000 (APC)
Diabetes Type I
1 in 500
Diabetes Type II
1-7% adults
Schizophrenia
1% lifetime risk
Chromosomal
Down's syndrome
1 in 625
"Modified from Science and Technology Committee (1995).
reaction (Findlay et aI., 1995), a multiplex oligonucleotide ligation assay for 30
common CF mutations accounting for >96% of CF carriers (Eggerding et aZ.,
1995), and whole genome preamplification allowing secondary amplification of
several DNA sequences from single cells (Zhang et aI., 1992; Kristjansson et aI.,
1994). For chn;)mosomal analysis, comparative genome hybridization (CGH)
(Kallioniemi et aI., 1992) may be possible by generating a probe from single
cells by whole genome amplification. This would allow karyotype analysis by
hybridizing a mixture of probes from the embryo cell and a normal cell to a
normal metaphase spread. Computer analysis of the relative strength of the
hybridization signals then allows identification of regions over or under represented in the test sample e.g. trisomies or monsomies. Alternatively, again using
computer-assisted analysis, analysis of all 23 pairs of chromosomes using
chromosome specific paint probes labelled with different fiuorochromes has
recently been achieved (Speicher et aI., 1996). If possible at the single cell level,
148
Preimplantation genetic diagnosis
this would enable screening for any translocations as well as aneuploidy and
some other structural abnormalities.
Single cell genetic analysis is already possible for many of the prevalent
inherited single gene defects in the UK (Table IV). Now because preimplantation
genetic diagnosis does not involve terminating established pregnancies, attention
is focussing on the potential for screening late onset diseases and other medical
conditions which may put the mother or baby at risk. With late onset disease,
individuals are often only affected after they have had children. If they know
family members have been affected they may not wish to know their own carrier
status but want to prevent their children inheriting the defect. One relatively
common example is Huntington's chorea. In these cases, termination of an
otherwise healthy fetus would be a difficult choice. Alternatively it is theoretically
possible to offer preimplantation genetic diagnosis without informing the parents
of their carrier status (Schulman et al., 1996). Screening for dominant single
gene defects which predispose carriers to familial forms of breast and colon
cancer is also being considered. In many of these cases, the penetration of the
gene defect is high and affected individuals will almost certainly develop tumours
in later life. In these conditions, even radical surgery may not completely
eliminate the risk of other malignancies associated with the loss of tumour
supressor activity. Beyond these conditions, however, screening for genetic
contributions to common diseases with a significant environmental component
is unlikely to be effective and raises serious ethical issues.
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