Molecular Human Reproduction, Vol. 8, No. 12, 1136-1143,
December 2002
© 2002 European Society of Human Reproduction and Embryology
Diagnosing inherited disease |
Preimplantation genetic diagnosis for ß-thalassaemia using sequencing of single cell PCR products to detect mutations and polymorphic loci
1 Department of Obstetrics and Gynaecology, University of Adelaide, The Queen Elizabeth Hospital, Woodville, 5011, South Australia, 2 Department of Molecular Pathology, Institute of Medical and Veterinary Science, Frome Road, Adelaide, 5000, South Australia and 3 Repromed, 4th Floor, Queen Elizabeth Hospital, Woodville, 5011, South Australia, Australia
Abstract
In order to carry out preimplantation genetic diagnosis (PGD) for ß-thalassaemia, we have applied direct sequencing of single cell PCR products to detect mutations and polymorphic loci within the ß-globin gene. Conventional duplex PCR was used to amplify two regions of the ß-globin gene with an amplification efficiency of 79% for blastomeres. Sequencing data were obtained for 100% of amplified products, with 12% having confirmed allele drop-out (ADO). A double ADO event was observed at least twice, confirming the real risk of such an event during PGD. In one couple, the presence of a polymorphism linked to the female partner's mutation enabled us to eliminate the risk of misdiagnosis due to double ADO without having to amplify both mutations within the same PCR product. We present here the data from eight clinical PGD cycles for three couples resulting in a singleton pregnancy and a twin pregnancy with all babies confirmed to be free from ß-thalassaemia (major).
allele drop-out/preimplantation genetic diagnosis/sequencing/single cell PCR/ß-thalassaemia
Introduction
Preimplantation genetic diagnosis (PGD) has been used for over a decade to allow couples to test for specific genetic conditions in the early embryo prior to the selection of embryos for transfer to the uterus to establish pregnancy. The use of IVF to create the embryos prior to embryo biopsy and testing is required, and an important consideration to justify the use of IVF for fertile couples is the accuracy of the diagnosis that is made. There have been four misdiagnoses reported by the ESHRE consortium (Geraedts et al., 2002
). Specifically, a misdiagnosis for ß-thalassaemia was reported recently (Palmer et al., 2002).
An analysis of the genotype of embryos consists of two steps, the PCR amplification of the locus or loci of interest followed by the analysis of the PCR products obtained to determine their genotype. Amplification methods for many diseases have been reported in the literature; these include cystic fibrosis (Handyside et al., 1992), Duchenne muscular dystrophy (Hussey et al., 1999; Ray et al., 2001), thalassaemias (Verlinsky and Kuliev, 2000), inherited cancer (Ao et al., 1998), Tay–Sachs disease (Sermon et al., 1995), Lesch–Nyhan (Ray et al., 1999), trisomy detection (Findlay et al., 1995) and Fanconi's anaemia with concomitant HLA typing (Verlinsky et al., 2001). A large number of methods for determining the genotype of the amplified PCR products have also been described. However, all methods have technical problems and/or errors associated with them.
With an estimated 269 million people affected by globin gene disorders worldwide, the thalassaemias are the most common human monogenic disorders in the world (Angastiniotis and Modell, 1998). ß-Thalassaemia is an autosomal recessive blood disorder which is characterized by an absence or a reduction in ß-globin chain synthesis and which displays phenotypic variation dependent upon the nature of the mutations involved (Kazazian, 1990). More than 100 different mutations located within the ß-globin gene on chromosome 11 are currently recognized throughout the world as causes for ß-thalassaemia (Kazazian, 1990).
Patients with two ß-thalassaemia null mutations have thalassaemia major and develop a lifelong transfusion-dependent anaemia (Webster and Lammi, 1994) which necessitates monthly blood transfusions and daily iron chelation therapy for life, to prevent serious physiological complications including cardiac failure and death.
In order to overcome the enormous burden that is placed not only on individuals with ß-thalassaemia and their families but also on society, ß-thalassaemia prevention programmes have been established and are active throughout the world. Prenatal diagnostic testing programmes exist so that chorionic villus sampling (CVS) and amniocentesis, followed by termination of affected pregnancies, are commonplace. Despite the success of these programmes, the large decrease in birth rates of affected babies translates to an alarmingly high abortion rate, which is unsuitable for many couples. For those who view non-use of embryos differently to termination of pregnancy, PGD may be more ethically acceptable.
However, one of the problems associated with PGD, not only for ß-thalassaemia but also for other diseases, is the possibility of misdiagnosis due to allele drop-out (ADO). ADO is an extreme form of preferential amplification where one allele of a pair of alleles fails to amplify or be detected. For hemizygous loci this phenomenon causes the complete absence of a PCR product. Thus, the highest accuracy for PGD can only be achieved when the diagnosis is reversed, that is the absence of the mutation is not sufficient for embryo transfer; instead, proof must be obtained that the embryo has inherited the chromosome(s) not carrying the mutation(s).
PGD methods for ß-thalassaemia published thus far, use either restriction enzyme digestion methods (Kuliev et al., 1998; De Rycke et al., 2001; Chamayou et al., 2002) or denaturing gradient gel electrophoresis (DGGE) for performing the mutation analysis (Kanavakis et al., 1999; Vrettou et al., 1999; Palmer et al., 2002). We consider direct sequencing for mutation detection to be a more accurate and simpler method and we have applied direct sequencing to PGD for ß-thalassaemia for both mutation and polymorphic marker detection. The presence of two nucleotides at one position in the sequenced products demonstrates the presence of two PCR products and thus confirms that ADO has not occurred. This results in an increased confidence in diagnosis.
Materials and methods
Patient data
Three couples known to be carriers of ß-thalassaemia requested PGD. Couple A had unproven (but assumed) fertility. The female partner was a compound heterozygote carrying the codon 39 (C to T transition at the first base of codon 39) and intervening sequence (IVS) I-6 (T to C transition at nucleotide 6 of the IVS I region) mutations. She was classified as having thalassaemia intermedia and a stabilized haemoglobin level of 7. Her medical history showed that she had been treated very conservatively with few blood transfusions. The male partner was a carrier for the IVS I-110 (G to A transition at nucleotide 110 of the IVS I region) mutation and had normal semen parameters (World Health Organization, 1999). The female was heterozygous for the codon 2 polymorphism (C/T) with the T linked to the IVS I-6 mutation. For this couple, each embryo would inherit a mutation from the female partner, therefore every embryo was an obligate heterozygote. Embryos lacking the IVS I-110 mutation would be suitable for transfer. The couple chose not to transfer embryos with the potentially milder genotype IVS I-6/IVS I-110.
Couple B presented to our unit with 6 months of infertility. Upon examination severe male factor infertility was diagnosed [sperm: 25x106/ml, 13% progressive motility and 7% normal forms (our unit reference value is 20% normal forms)]. The couple chose to have conventional IVF with ICSI and conceived twins on their first cycle. At 11 weeks, a CVS was performed and one twin was found to be carrying both mutations for ß-thalassaemia. The twin pregnancy was successfully reduced with the healthy twin delivered at term without complications. After weighing up the possible detrimental effects of the embryo biopsy procedure against the unhappy outcome of a termination of pregnancy (especially for a singleton pregnancy), they decided to have PGD for their second pregnancy. The female partner was a carrier for the IVS II-745 mutation (C to G transversion at nucleotide 745 of the IVS II region) and the male partner was a carrier for the IVS I-110 mutation. The female partner was heterozygous for the codon 2 polymorphism (C/T) at this position with the T allele linked to the IVS II-745 mutation. Her partner was homozygous C at this polymorphic locus, making this polymorphism 100% genetically informative for PGD purposes (i.e. the presence of the T allele would equate with the presence of the IVS II-745 mutation in their offspring). The female partner also carried the IVS II-666 polymorphism (T to C transition) with the C linked to the IVS II-745 mutation. Similarly, this couple was also informative for PGD for the CAP+20 polymorphism (results not shown).
Couple C presented with 2 years of infertility. Severe male factor infertility was diagnosed (sperm: 11.7x106/ml, 51% progressive motility and 2% normal forms) and ICSI/IVF with PGD was recommended. The female partner was a carrier for the codon 39 mutation and the male partner carried the IVS I-110 mutation.
Isolation and preparation of single lymphocytes
Single peripheral lymphocytes were isolated from whole blood and DNA was extracted exactly as previously described (Hussey et al., 1999).
Embryo biopsy and isolation of single blastomeres
Retrieved oocytes were fertilized by ICSI and the embryos were cultured until the morning of day 3 (usually, but not always, embryos have reached the 5–10-cell stage by this time) when embryo biopsy was performed. A hole was made in the zona pellucida using a FertilaseTM system (Medical Technologies Montreux, Switzerland). A 30 µm biopsy pipette was inserted through the hole to remove a nucleated blastomere by aspiration. The blastomere was washed three times in 1xPCR buffer (50 mmol/l KCl, 10 mmol/l Tris–HCl, pH 8.3) supplemented with 0.01% PVP using a freshly pulled Pasteur pipette. The blastomere was observed under a dissecting microscope as it was placed into a 0.5 ml PCR tube. Once the blastomere was in the tube, the Pasteur pipette was removed and the remaining buffer was placed into another PCR tube to act as a negative control. On rare occasions no buffer remained and therefore buffer from the last wash drop was aspirated and used as a negative control. Embryo transfer was carried out on day 4 or 5.
First round duplex PCR procedure
The first round nested PCR was carried out using a modification of a published method (Kuliev et al., 1998). Two pairs of outer primers (Table I, Figure 1) were used to amplify the IVS I and IVS II regions of the ß-globin gene simultaneously. Single cell lysis was carried out by a published method (Cui et al., 1989), with 5 µl of lysis buffer (200 mmol/l KOH, 50 mmol/l dithiothreitol) for 10 min at 65°C followed by neutralization with 5 µl of neutralization buffer (300 mmol/l KCl, 900 mmol/l Tris–HCl, pH 8.3, 200 mmol/l HCl). Negative and positive controls were also lysed. Lysates were centrifuged briefly and placed immediately back on ice or frozen at –20°C until use. To each lysed and neutralized cell (10 µl), a 40 µl aliquot of master mix (10 µl of 10% glycerol or purified water, 3 µl of 25 mmol/l MgCl2, 8 µl of 10 mmol/l dNTP mix (2.5 mmol/l each), 20 pmol of each primer (Table I), (0.3 µl) 1 IU Taq polymerase (Perkin Elmer, Norwalk, CT, USA) and PCR grade water (Biotech International, Perth, WA, Australia) to a volume of 50 µl was added and overlayed with 50 µl of mineral oil. The tubes were placed into a thermocycler (Minicycler; MJ Research, Cambridge, MA, USA) and cycled for an initial denaturation at 96°C for 5 min followed by 28 cycles of 96°C for 30 s, 45°C for 1 min, 72°C for 1 min proceeded by a final extension of 72°C for 5 min.
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Second round PCR procedure
Individual second round PCR were performed for nested and heminested IVS I and IVS II regions using inner primers (Table II
). A 3.5 µl aliquot from the first round PCR reaction was used to seed the individual second round PCR reactions. The aliquot was added to 5 µl 10xPCR buffer (500 mmol/l KCl, 100 mmol/l Tris–HCl, pH 8.3), 3 µl 25 mmol/l MgCl2, 4 µl of 10 mmol/l dNTP mix (2.5 mmol/l each), 20 pmol each of the forward and reverse primers (0.3 µl) 1 IU Taq polymerase and ultra-pure water to a volume of 50 µl. After an overlayer of 50 µl of mineral oil, tubes were placed in a thermocycler. Following an initial denaturation for 4 min at 96°C, the nested IVS I and IVS II amplification consisted of 25 cycles of amplification involving 94°C for 45 s, 52°C for 60 s and 72°C for 60 s with a final extension of 72°C for 5 min. Cycling conditions for heminested IVS I amplification consisted of 20 cycles of amplification involving an initial denaturation of 96°C for 5 min, 94°C for 45 s, 62°C for 45 s and 72°C for 60 s followed by a final extension of 72°C for 5 min.
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PCR products were electrophoresed on 2% agarose gels in 0.5xTBE (Tris, borate, EDTA) prestained with ethidium bromide and photographed.
Sequencing procedure
Following gel electrophoresis confirming successful amplification of the correct size product, the remaining PCR product (35 µl) was purified using a BRESAspinTM PCR Purification Kit (Geneworks, Adelaide, South Australia). Purified PCR products were sequenced using an ABI PRISMTM BigDye Terminator Cycle Sequencing Ready Reaction Kit protocol (Perkin Elmer Applied Biosystems). Briefly, to a 0.5 ml PCR tube 30–100 pg of DNA, 3 pmol of reverse primer, 4 µl Terminator Ready Reaction Mix was added and made up to a final volume of 10 µl with ultra-pure water. The addition of too much template results in sequencing runs being too short (i.e. sequence data are lost before reaching the end of the PCR product). The addition of too little template results in faint signal that cannot be detected above the background. In order to determine correctly how much purified product as template to add to the sequencing reaction, we electrophoresed a portion of our sample, through 2% agarose minigels prestained with ethidium bromide, alongside a standard of known concentration that had previously been successfully sequenced. The amount of template added to the sequencing reaction was estimated by comparison with the known amount contained in the standard. The sequencing products were cycled in a Hot Bonnet PCR thermocycler (Minicycler) for an initial denaturation for 30 s at 96°C, followed by 25 cycles of 96°C for 10 s, 53°C for 5 s and 60°C for 4 min.
The extension products were precipitated using isopropanol, which is the preferred method as the resulting products can be analysed on both gel and capillary instruments. The slight disadvantage of being unable to see the DNA pellet was circumvented by the addition of a small amount of glycogen (5–10 µg). The glycogen pellet, however, needed to be broken up completely during the washing steps. To the sequencing reaction was added 40 µl of 75% isopropanol and this was left at room temperature for 
15 min but not overnight. Samples were centrifuged at 12 000 g for 20 min and the supernatant removed and discarded. To the pellet, 250 µl of 75% isopropanol was added and briefly vortexed. After centrifugation for 5 min at 12 000 g, the supernatant was aspirated using a vacuum water pump and the sample was dried gently.
Samples were analysed on the ABI sequencers (373, 377 or 3700 Perkin Elmer Applied Biosystems) as per the manufacturer's instructions.
Results
Pre-clinical workup
Slight modifications to the nested PCR protocol published by Kuliev et al. (1998) were made to give the same quality product with fewer cycles of amplification. It was our policy to sort single lymphocytes from both partners of every couple to confirm that the test works on the couple's own single cells before proceeding with PGD. In total, from these three couples, 208 single lymphocytes were amplified simultaneously at the IVS I and/or the IVS II region of the ß-globin gene. From the 208 lymphocytes, 188 (90%) produced a product(s) of the correct size. The remaining 20 lymphocytes failed to amplify at either locus. Of these 188 amplification products, 60 were purified and sequenced and two alleles were clearly visible in 59 (i.e. 98% were heterozygous). We observed no contamination resulting in the presence of polymorphisms or mutations which were absent in the individual from whom the cell was taken. The ADO rate using our method for lymphocytes that amplified was therefore ~2%.
Since the codon 39 mutation is located 19 bp from the fully nested reverse primer for IVS I (N2), and was best detected using a heminested PCR we standardized our protocol for all mutations to use the heminested instead of the fully nested second round reaction for the IVS I region. For clinical cases where one of the patients carried the codon 39 mutation (couples A and C), we performed the heminested second round PCR reaction instead of the fully nested reaction. Furthermore for cycles A1–A3, B1 and B2 we sequenced in both directions (forward and reverse); however, when we had become experienced we sequenced in only a single direction.
Clinical cycles of PGD
The results of the eight clinical PGD cycles performed for ß-thalassaemia are shown in Table III and Figure 2. The strategy used to detect ADO in the embryos from couple B is shown in Figure 3. This strategy avoids the transfer of an affected embryo in the event of a double ADO event.
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Couple A had three stimulated cycles of PGD with no pregnancy. Follicular fluid from the female partner of couple A was very yellow in colour, suggestive of haemosiderin and indicative of iron overloading. Treatment for this couple was suspended after three cycles when pulmonary hypertension was diagnosed.
Couple B had embryos frozen singularly after their successful first conventional IVF/ICSI cycle. For their first PGD cycle it was decided that embryos would be thawed until three survived. 4-cell embryos were thawed and cultured overnight, and were thus day 4 embryos which were considered to be as if they were day 3 embryos. Two 8-cell embryos were thawed on the morning of day 3, allowed to equilibrate for 1 h and then biopsied. The 4-cell embryo failed to divide after biopsy, whereas the two 8-cell embryos divided by day 4. Therefore these were transferred and a pregnancy ensued. At 7 weeks the patient had some bleeding and a scan showed a single sac but no fetal heart beat. A miscarriage resulted.
Since the 4-cell embryo fared the worst after thawing, we could not be sure how well any of the other 4-cell embryos in storage would survive thawing, culturing and biopsy. The embryo quality obtained after the first stimulated cycle 3 years previously was excellent. As there were only 4-cell embryos left in storage, it was recommended that the couple undergo a second stimulated cycle, this time with PGD. No pregnancy resulted and the couple elected to repeat the testing of frozen embryos from their first cycle. Three embryos were thawed and tested. The embryo quality of two of these was too poor for transfer. The other embryo was of good quality but had both ß-thalassaemia mutations and thus would develop into a child with thalassaemia. No embryo transfer was carried out.
After this disappointment, the couple chose to have a frozen embryo transfer of the embryo from the second stimulated cycle which was at 50% risk of thalassaemia since we had shown that it carried at least one mutation, despite the fact that they had (three) frozen embryos from the first cycle at 25% risk. The couple was informed that this went against our recommendations and they agreed to have a CVS. No pregnancy resulted and the couple booked for a third stimulated cycle with PGD. This one was successful with a single fetal heart beat at scan (7.5 weeks). A CVS was performed at 11 weeks confirming that the fetus was free of ß-thalassaemia, that it had inherited the IVS II-745 but not the IVS I-110 mutation and had an apparently normal male karyotype.
Couple C had one stimulated cycle and two embryos transferred, an ongoing twin pregnancy resulted. The couple decided against prenatal diagnosis. An analysis of the cord bloods was performed on the twins at birth. The genotypes were the same as was diagnosed for the two embryos that were transferred.
In total, 53 blastomeres were removed from 44 embryos. Each embryo had a single cell removed except for nine embryos which had a second cell removed. The reasons for this were that the first biopsied cell lysed during biopsy or while being removed from the biopsy pipette (eight embryos) or that the nucleus was not visible after biopsy (one embryo of extremely poor quality). A single blastomere from 43 of the 44 embryos was analysed and for virtually all (98%) blastomeres analysed, a negative control was also analysed.
Of the 43 blastomeres successfully removed from the embryo, 35 produced PCR amplification products. This gives an amplification efficiency of ~81%. The first round was always a duplex PCR for both the IVS I and IVS II regions even when a mutation within the IVS II region was not carried by the couple. The PCR products from these 35 blastomeres were analysed at the IVS I region by sequencing to determine their genotype and those from couple B were also analysed at the IVS II region. In addition, two negative controls produced PCR products and the unique nature of these two products is discussed later. Thus the total number of PCR products sequenced from both loci was 52. All of these gave a readable sequence (100% success rate). For IVS I region PCR products (the diagnostic locus), six (12%) PCR products had confirmed ADO. For two of these (embryos B2, 2a and C1, 6a) we cannot be sure if ADO or separation of the two chromosomes occurred (see Discussion). A further five PCR products could not be determined for the presence of ADO as the absence of two different alleles was consistent with the embryo's potential genotype, and at least some of these would be expected to be true homozygous unaffected embryos. Furthermore one of these (embryo C1, 11) gave rise to a homozygous unaffected baby. There were two pregnancies from six fresh cycles (30%) and one pregnancy from two frozen cycles (50%). This is similar to our overall pregnancy rate for disorders diagnosed by PCR of 38% per embryo transfer for the year 2000.
Discussion
The use of sequencing to detect sequence variation within PCR products generated from the amplification of blastomeres obtained after embryo biopsy has proven to be extremely useful. The main advantage of sequencing PCR products is that the same method can be applied to virtually any sequence regardless of the gene, locus or DNA change. The main exception to this is the sequencing of PCR products containing repeated sequences. The presence of two nucleotides at one position in the sequenced products demonstrates the presence of two PCR products and thus confirms the presence of two alleles. When only a single nucleotide is present, when two were expected, ADO is detected and a serious misdiagnosis can be avoided.
Couples A and C were carrying mutations within the same amplified PCR product and thus the occurrence of ADO would cause the transfer of a carrier embryo when it was diagnosed as homozygous unaffected but not the transfer of an affected embryo. Therefore a serious misdiagnosis would not occur due to ADO alone but due to contamination. Couple B on the other hand carried mutations in separate regions of the ß-globin gene. There were therefore two options; one was to redesign the PCR so that it covered the whole 1.5 kb region of the gene to encompass both mutations in the same amplification product. This required the optimization of new primers and, due to the length of the PCR product amplified, the efficiency of the reaction may be susceptible to slight variations in conditions. Nevertheless at least one group has reported using this method (Cram et al., 2000). The second option was to take advantage of the presence of polymorphisms within the ß-globin gene. The IVS II-745 mutation is linked (Orkin et al., 1982) to a number of polymorphisms including codon 2, IVS II-666 and CAP+20 (position 20, 5' untranslated region). Two of these (codon 2 and CAP+20) are located within the region amplified by our IVS I region primer pair.
From sequencing of DNA from couple B, we knew that the codon 2 polymorphism (C/T) was fully informative for PGD in this couple because the female was heterozygous with the T linked to the IVS II-745 mutation and her partner did not carry the T polymorphism at this position. Had the codon 2 polymorphism not been fully informative, it would have been simple to analyse the CAP+20 polymorphism instead (fully informative in couple B for PGD). The diagnosis strategy for this couple was that a blastomere producing a PCR product that did not contain either the codon 2 `T' polymorphism nor the IVS I-110 mutation could be sure to carry at least one normal copy of the ß-globin gene. Couple B decided that they would not proceed with PGD unless it was 99% accurate. That is, they would rely solely on CVS if the problem of misdiagnosis due to double ADO could not be addressed. This was because they had concerns about safety of the embryo biopsy procedure. Our sequencing strategy enabled us to offer PGD to this couple at the same level of accuracy as that for couples A and C.
We report here three pregnancies from six fresh IVF/PGD and two frozen PGD cycles. The first pregnancy (couple B) miscarried at 7.5 weeks gestation and unfortunately no products of conception were obtained to confirm the diagnosis. Later this couple conceived a singleton pregnancy, which was tested by CVS and shown to be a carrier for the IVS II-745 mutation. It is not possible to say which embryo gave rise to the singleton birth, as it could be embryo 11 or embryo 1 with ADO of the IVS II-745 allele. Couple C gave birth to healthy twins by Caesarean section at 36 weeks 5 days gestation. This couple declined prenatal diagnosis and a cord blood analysis was performed at birth. One twin was confirmed as homozygous unaffected and the other twin as a carrier for the IVS I-110 mutation. This result is completely consistent with the diagnosis obtained for the two embryos that were transferred. Couple A was forced to withdraw for medical reasons before a pregnancy was achieved.
Currently mutation and polymorphism detection systems used by PGD centres around the world include restriction endonuclease digestion either with or without primer modification to create a restriction enzyme site. This method was not favoured by us as restriction enzyme digestion can fail and in many diagnostic strategies (De Rycke et al., 2001; Chamayou et al., 2002) this would give a false normal result and allow the transfer of an affected embryo. In addition, some background cutting (i.e. non specificity) may occur with restriction enzymes, especially when modified primers are used, as the Taq polymerase can non-specifically incorporate a mismatched base into the diagnostic position. The best option for us was to choose direct sequencing of the single cell PCR products to detect the mutations and polymorphisms and hence to detect ADO. The use of SNUPE (single nucleotide primer extension) was considered but rejected as this type of minisequencing is only able to look at a single nucleotide position. Furthermore it requires the addition of more than one primer and optimization thereof if more than one site is to be looked at.
As there is only a single copy of each of the two alleles present in a single cell, both need to be amplified for PGD to be accurate. The accuracy of PGD by PCR relies heavily on the fidelity of the amplification process. The occurrence of PCR-induced errors is unavoidable and can result in a severe misdiagnosis. The techniques of DGGE and single-stranded confirmation polymorphism for mutation detection (Vrettou et al., 1999) are unable to pinpoint the exact type and location of the change in the DNA that results in a deviation from the (normal) expected pattern. For these reasons, direct sequencing provides more information. Mutation detection techniques that rely on annealing differences between mutant and normal allele types [such as allele specific oligonucleotides, amplification refractory mutation system, reverse dot blot (Sheardown et al., 1992; Chamayou et al., 2002)] require different conditions for each mutation and are very sensitive to small changes in the temperature and to changes in the stringency of the annealing/hybridization step. Sequencing on the other hand uses standard conditions for all templates and does not appear to be very sensitive to changes in day-to-day use. The accurate quantification of the template DNA is however very critical but can be easily controlled by a direct comparison with previously sequenced samples or a control template.
Compared with the ABI 373, which required overnight runs, the new ABI 3700 capillary machine is able to produce results within 4 h. The cost of sequencing is Australian $11.00 (equivalent to US$ 6.60 or 19.60 euros) per sample. The disadvantage of cost is far outweighed by the convenience and accuracy of the results obtained. It is always advisable to sequence the parental DNA prior to PGD to confirm that the mutations present can be detected using the sequencing strategy.
Of concern was the fact that two blastomere wash negative controls produced amplification products. These were for embryo 2 of the B2 (033) cycle and embryo 6 of the C1 (045) cycle (Table III
and Figure 2). When placing the blastomere into the PCR tube, we attempt to visualize it. In these two instances the cell was not visualized going into the tube. Therefore we could not say which amplification band corresponded to the cell (if any). As amplification products were produced from both tubes, this can be interpreted in two ways. One was that the cell was placed into the first tube and that there was contamination resulting in a false positive band in the second tube (or vice versa). The second interpretation was that the cell lysed in the pipette and that it is possible that one chromosome 11 went into the first tube and that the second chromosome 11 went into the second tube. The sequencing results are consistent with either option so it is not possible to say which of the two options is correct. In the case of the B2 (033) cycle, the couple elected to transfer this embryo but a pregnancy did not ensue. In the case of the C1 (045) cycle, the couple chose not to transfer that embryo as there were other good quality embryos.
We have taken advantage of this technique of fractionating a single cell into several aliquots with a view to determine the haplotype of a chromosome or region(s) of a chromosome. We have called this technique HACS (Haplotyping After Chromosome Separation) and have shown that it is possible to separate chromosome pairs into different tubes and obtain information about the genes or markers on each of the chromosomes. In this way it is possible to obtain genetic information about a chromosome when this information is masked by the genes or markers on the other chromosome (unpublished data).
We report here the first babies born as a result of using sequencing to analyse mutations and polymorphic loci for PGD for ß-thalassaemia. We believe that direct sequencing provides the highest accuracy diagnosis available after single cell PCR amplification.
Acknowledgements
We wish to thank Svetlana Rechitsky, Charles Strom and Yury Verlinsky for providing assistance, information and their generous gift of primers prior to publication. We would also like to thank David Froiland for single cell sorting and Arthur Mangos from the Sequence Service, IMVS. This work was supported by an NH&MRC grant to N.D.H. and C.D.M. #991345 (GN ID 9938254).
Notes
4 To whom correspondence should be addressed. E-mail: nicole.hussey{at}adelaide.edu.au 
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Submitted on June 26, 2002; accepted on September 27, 2002.
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