Molecular Human Reproduction, Vol. 7, No. 9, 895-901,
September 2001
© 2001 European Society of Human Reproduction and Embryology
Reproductive genetics |
The development of preimplantation genetic diagnosis for myotonic dystrophy using multiplex fluorescent polymerase chain reaction and its clinical application
1 Department of Obstetrics and Gynecology, 2 Department of Experimental Medicine and 3 Department of Human Genetics, McGill University, Royal Victoria Hospital, Montreal, Canada, H3A 1A1
Abstract
Preimplantation genetic diagnoses (PGD) for single gene defects require considerable time and resources for the standardization of polymerase chain reactions that are rapid, sensitive and reliable. Developing tests for the trinucleotide repeat diseases, where the expansion of unstable repeats produces the phenotypes, are particularly complex. One of these disorders is myotonic dystrophy where, at present, diagnosis at the single cell level relies on the detection of the normal alleles from both the affected and unaffected parent. The incorporation of short tandem repeat polymorphisms in the assay can give additional information to improve the accuracy of diagnosis. We have developed a multiplex fluorescent reaction for myotonic dystrophy and one of two closely mapped, highly heterozygous, short tandem repeats (D19S219 and D19S559) on chromosome 19 to reduce the possibility of misdiagnosis due to contamination, act as a control for allelic drop-out and maximize the number of embryos genotyped. This protocol was designed as a general diagnosis for myotonic dystrophy, using the most informative of the two polymorphisms for each couple. Subsequently this approach was used in a PGD treatment cycle.
multiplex fluorescent PCR/myotonic dystrophy/preimplantation genetic diagnosis/short tandem repeat polymorphism
Introduction
The first clinical application of preimplantation genetic diagnosis (PGD) for single gene defects was for cystic fibrosis in 1992 (Handyside et al., 1992
) and although over 5000 single gene defects have been identified (McKusick, 1998), PGD has only been applied to a few of these disorders. The development of a new test at the single cell level is challenging and involves intensive time and resources to standardize a polymerase chain reaction (PCR) protocol unique for the specific mutation of interest. The PGD must be possible within 1 or 2 days to allow the transfer of biopsied IVF embryos within the time window for successful implantation. It must also be sensitive to give the highest efficiency of amplification from the two copies of the template found in a diploid cell, therefore allowing the maximum number of diagnosed embryos. Finally, the diagnosis given by the test must be accurate to ensure that only unaffected embryos are transferred. Reliable and efficient diagnosis maximizes the selection of good quality unaffected embryos available for embryo transfer and thereby, hopefully, the success of the treatment. This is even more evident when performing PGD for an autosomal dominant condition where, by the rules of Mendelian transmission, 50% of embryos will, theoretically, be unaffected.
Among genetic conditions are a group of trinucleotide repeat disorders, of which there are currently 14 (Cummings and Zoghbi, 2000) that cause a progressive pathological phenotype when there is an expansion of the repeat. The high CG content of most of these repeats makes the development of PGD protocols particularly challenging and clinical PGD has only been reported for three of these conditions, namely, myotonic dystrophy (Sermon et al., 1997, 1998a), fragile X syndrome (Sermon et al., 1999) and Huntington's disease (Sermon et al., 1998b).
Myotonic dystrophy (DM) is the most common form of adult onset muscular dystrophy. It is a dominantly transmitted multisystemic disorder with both the phenotypic expression and the age of onset being variable. DM symptoms usually present in the third or fourth decade of life and are progressive, often leading to significant disability. A second, more severe, congenital form is associated with an increased perinatal mortality rate. The unstable CTG repeat is located in the 3' untranslated region (UTR) of the myotonic dystrophy protein kinase (DMPK) gene located on chromosome 19q (Brook et al., 1992; Mahadevan et al., 1992). The CTG repeat sequence in the DM gene tends to increase in size in affected individuals, and the severity of the disease is correlated with the length of the repeat expansion (Buxton et al., 1992; Harley et al., 1993). Unaffected individuals have 5–37 repeat units, mildly affected 50–150, adult onset patients 100–1000 and congenital cases have 1500–6000 repeat units (Brook et al., 1992). In general there is a progressively earlier onset, and increased severity of DM, as the condition is transmitted to successive generations of a family (Harper et al., 1992). This phenomenon is called anticipation.
At present, the PCR protocols for DM are unable to amplify the expanded repeat at the single cell level, so the diagnosis depends on the detection of the healthy allele from the affected parent. The CTG repeat unit is highly heterogeneous in the general population and this has been used in the development of PGD for DM (Sermon et al., 1997, 1998a). In this case, candidate couples for PGD must be informative, i.e. the healthy allele of the affected parent should be different from the alleles of the unaffected parent. This limits diagnosis and leaves open a possibility for misdiagnosis from either DNA contamination or allelic drop-out (ADO): the non-amplification of one allele in a heterozygous sample (Findlay et al., 1995; Ray and Handyside, 1996). According to a recent paper which devised a model for controlling the rate of misdiagnosis in PGD (Lewis et al., 2001), the chance of an affected embryo being classified as unaffected, when diagnosing a dominant disorder, from the disease allele alone is 10.9%. However, additional information gained from testing the genotype of a linked marker reduces the possibility of replacing an affected embryo to 0.1%.
We describe here the development and standardization of a multiplex fluorescent PCR for DM and one of two closely mapped, highly heterozygous, short tandem repeats (STR) on chromosome 19 (Table I) (Gyapay et al., 1994). We believe a multiplex approach is an improvement on presently available tests in reducing the risk of replacing an affected embryo. This method can also maximize the number of good quality unaffected embryos available for selection at transfer by increasing the number of embryos on which a genotype can be assigned. It was designed as a general PGD for DM, using the most informative of the two STR for each particular couple. The work was approved by the Institutional Review Board of the Hospital. Once developed, the technique was then applied for PGD for DM for a particular couple.
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Materials and methods
Isolation of research cells
The isolation of single lymphocytes and research blastomeres, using the alkaline lysis buffer system, was as previously described (Blake et al., 1999). Each sample was kept at –80°C for at least 20 min before processing and then the cells were lysed by incubating at 65°C for 15 min.
Genetic analysis of single cells
A heminested approach was used to amplify the CTG repeat in the DMPK 3'UTR using three primer sequences (Table I
). DNA primers were also designed in a heminested PCR to amplify D19S219 and D19S559 STR (Table I) from polymorphic loci on chromosome 19. The forward primers (inner forward in the case of D19S559) in each set were labelled with CY5 fluorescent dye and stored at a stock concentration of 40 µmol/l in molecular biology grade water (Sigma) at –20°C.
Reaction mastermixes for the first round of PCR were in a total volume of 50 µl and included a final concentration of 5% dimethylsulphoxide (DMSO), 1xneutralization buffer [900 mmol/l Tris–HCl (pH 8.3), 300 mmol/l KCl, 200 mmol/l HCl], 1xpotassium free PCR buffer [25 mmol/l MgCl2, 100 mmol/l Tris–HCl (pH 8.3)], 100 µmol/l of each dNTP, 1 unit of Taq polymerase (Qiagen, Mississauga, Ontario, Canada), and 0.4 µmol/l of DMF and DMOR primers and 0.4 µmol/l of either 219F and 219OR or 559OF and 559R. The first round of PCR was carried out on a PTC-200 thermocycler (MJ Research, Watertown, MA, USA) using the following programme: 5 min denaturation at 96°C, followed by 15 cycles of 1 min at 96°C, 45 s at 60°C and 1 min at 72°C, followed by 5 min at 72°C.
For the second round of DNA amplification, 2 µl of the first round products were added to reaction mixes in a total volume of 30 µl with a final concentration of 1xPCR buffer [200 mmol/l Tris–HCl (pH 8.4), 500 mmol/l KCl, 15 mmol/l MgCl2], 100 µmol/l of each dNTP, 1 unit of Taq polymerase (Qiagen) and either 0.4 µmol/l of DM primers (DMF and DMR), 0.4 µmol/l of D19S219 primers (219F and 219R) or 0.4 µmol/l of D19S559 primers (559F and 559R). DM, D19S219 and D19S559 were amplified separately in the second round using the following conditions: 5 min denaturation at 96°C, followed by 10 cycles of 30 s at 96°C, 45 s at 62°C and 1 min at 70°C, then 15 cycles of 1 min at 94°C, 45 s at 62°C and 1 min at 70°C and finally 5 min at 72°C.
A 3 µl sample of the fluorescently labelled amplified DNA was mixed with 4 µl of loading dye and denatured for 3 min at 90°C prior to the samples being loaded on to a 9% bis/acrylamide denaturing gel and run on an ALF Automated DNA Sequencer (Amersham Pharmacia Biotech). DNA was visualized as fluorescent peaks using the instrument's packaged computer software. A fluorescently labelled 50–500 bp marker (Amersham Pharmacia Biotech) was loaded on to each gel and used to size peak fragments.
Patient description
A 32 year old asymptomatic woman presented at the McGill Reproductive Centre interested in PGD for DM. She had been diagnosed as having an expanded CTG repeat on one copy of her DMPK gene at the age of 18 and had previously undergone a termination of pregnancy after the fetus was found to be carrying an expanded DMPK allele. Genetic testing for DM using DNA from peripheral blood samples from the prospective couple was carried out. Both partners were found to be informative for their normal alleles. The affected female had a healthy repeat of five units and an expanded repeat of 444 units. Her husband was homozygous for 13 repeat units. This was verified for the normal alleles using the fluorescent PCR protocol described in this paper. From the two chromosome 19 STR available (Table I) the patients were found to be uninformative as a couple, for the D19S559 tetranucleotide STR, but informative as a couple for the D19S219 dinucleotide STR, even though each individual was homozygous for the D19S219 STR. Therefore the dinucleotide repeat was used for the PGD. After the patients were counselled on IVF and PGD and the possible outcomes, they consented to undergo an IVF treatment cycle.
IVF and embryo biopsy procedure
IVF treatment was performed by standard protocol as previously described using heparinized saline for flushing the follicles (Biljan et al., 1997). Oocytes were identified, washed in equilibrated IVF-20 medium (Scandinavian IVF Science, Gothenburg, Sweden), then incubated (under humidified conditions with 5% CO2) in organ culture dishes containing 0.6 ml equilibrated medium in the centre well. Oocytes were denuded ~1 h after collection and intracytoplasmic sperm injection (ICSI) was performed with the partner's spermatozoa, after a further hour of incubation, in HEPES-buffered medium (Gamete-20; Scandinavian IVF Science) drops. Injected oocytes were washed and placed in individual 20 µl G1.2 (Scandinavian IVF Science) drops under oil. Sixteen to 18 h later, injected oocytes were assessed for normal fertilization (presence of two pronuclei).
On the morning of biopsy (day 3 of embryo development), the number of cells in each embryo and its grade (Dean et al., 2000) was assessed. Prior to biopsy, each embryo was incubated for 5 min in Ca2+- and Mg2+-free media to allow decompaction of blastomeres. After this, the embryo was placed in a drop of HEPES-buffered medium (Gamete-20; Scandinavian IVF Science) and biopsied, as previously described (Ao and Handyside, 1995). After removal, each blastomere was washed through two drops of medium, the presence/absence of a nucleus noted and transferred to a 0.2 ml PCR tube containing 5 µl alkaline lysis buffer. For each embryo biopsied, a sample of media from the drop in which the blastomere(s) had been washed was placed in a PCR tube with 5 µl alkaline lysis buffer to act as a `blank' for that embryo. All samples were processed as for the research cells described earlier, including the pre-processing step at –80°C. After biopsy the embryo was washed through three 40 µl droplets of G2.2 media (Scandinavian IVF Science), and placed in a clean labelled 40 µl drop under oil. Each biopsied embryo was incubated in a separate dish.
Results
A reliable single cell PCR assay was developed for DM multiplexed with each of the STR. Initially, a singleplex reaction was designed for DM using 36 cycles. This gave an acceptable amplification rate from 218 single lymphocytes (91.2%) and a reasonable ADO rate (8.5%). This PCR protocol also worked for DM multiplexed with both D19S219 and D19S559 (data not shown). However, as the analysis print-outs from the DNA sequencer did not give completely flat background peaks in any of these single round PCR assays, there was concern that non-specific bands would cause problems in diagnosis, so this was not considered suitable for clinical PGD analysis. Therefore, a heminested approach was employed in a two round PCR protocol. After standardization of suitable PCR conditions, the protocol was tested on single lymphocytes. From 50 lymphocytes used for DM with D19S559, the amplification rate was 92% for both loci and ADO was 4.3% for DM and 6.7% for D19S559. From the 40 lymphocytes assayed for DM with D19S219, the amplification rate was 100% for both loci with 5% ADO for DM and 0% for D19S219.
The final standardization to verify the accuracy and efficiency of the PCR assay for DM multiplexed with D19S559 or D19S219 was performed using a blinded approach. A total of 132 samples for DM with D19S219 and 36 samples for DM with D19S559 were coded, such that the individual assigning diagnoses was not aware of the expected number of single lymphocyte samples from each of six donors or blanks. All donors were heterozygous for their DM and STR profiles so that ADO could be calculated. The profiles of the donors for DM and both of the STRs can be seen in Figure 1. The genuine allele for the D19S219 dinucleotide repeat is sized according to the largest fragment amplified and the smaller peaks seen in the profiles (see Figure 1b) are spurious DNA fragments called `stutter bands'. These represent a PCR artefact caused by the slipping of the Taq polymerase over the CA repeat unit, which generates amplified fragments that differ by exactly 2 bp in length (Walsh et al., 1996). The overall amplification rates were >90% and the rates of ADO were <10% (Table II). The accuracy of diagnosis is defined as the ability to detect the correct genotype of the original cell from the amplified products of PCR. If there was no amplification, this was counted as a failure of the PCR assay and not as a misdiagnosis. For DM with D19S559 there was 100% accuracy of diagnosis and for DM with D19S219 it was 99%. The reason this was not 100% was due to the inability to designate a genotype for one of the lymphocytes due to allele drop-out. One of the blanks amplified DM products of a size that matched the DM donor genotype from which the blank was collected.
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Forty-five blastomeres from 10 embryos donated for research from one patient, were analysed to test the accuracy and sensitivity of the PCR protocol in embryonic cells. The cells were collected, lysed and analysed the same day to reproduce the situation in a clinical PGD. The amplification rate for DM was 85% (38/45 cells) with 2.6% (1/38) ADO, and for D19S219 the amplification and ADO rates were 80% (36/45 cells) and 8.3% (3/36) respectively. None of the 10 blanks tested showed amplification of any product.
For the clinical case, 13 oocytes were collected, 11 were injected and 10 were fertilized. On day 3, it was possible to biopsy nine of these embryos of which two had 8 cells, one had 7 cells, two had 6 cells and four had 5 cells. If an embryo had 
7 cells, two blastomeres were biopsied, but if it had <7 cells, only one blastomere was removed. An example of the print-out of the PGD using fluorescent PCR can be seen in Figure 2. From the 12 blastomeres tested, the amplification rate for DM was 91.7% (11/12) with an ADO rate of 0% (confirmed in the spares) and amplification for the D19S219 STR was also 91.7% (11/12) but with ADO of 18.2% (2/11). None of the 10 blanks tested showed amplification of any product. Two embryos diagnosed as unaffected were transferred to the patient but unfortunately she did not conceive. After the clinical case, 22 blastomeres from non-transferred embryos were tested and the results of the PGD were verified (results not shown).
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Discussion
We have demonstrated an accurate and efficient fluorescent multiplex PCR for the PGD of myotonic dystrophy. This was developed with one of two closely mapped, highly heterozygous, short tandem repeats on chromosome 19. An example of the analysis obtained using this protocol can be seen in Figure 1. We can confidently determine the difference between one repeat unit (three base pairs) for DM (donor 5 in Figure 1) and the different profiles for both STRs. We believe that co-amplification, in a multiplex fluorescent PCR with a polymorphic marker, can improve DM PGD in several ways.
First, the possibility of misdiagnosis due to contamination is reduced. At present, PGD testing for informative DM couples depends on identification of the normal allele from the affected parent, as protocols are unable to amplify the expanded repeat at the single cell level. As the amplified region of the DM locus is a repeat, different individuals will show a particular profile after PCR for their normal DMPK alleles. This gives an internal control in the PGD assay, in that if PCR detects a particular DM CTG repeat in a blastomere, not found in either parent, the presence of contaminating DNA would be identified. However, incorporation of external contaminating DNA with the same normal repeat size as either parent would not be detected as contamination. If this was the same size as the normal allele from the affected parent, it would lead to misdiagnosis. As DM is a dominant condition, misdiagnosis may result in the transfer of an affected embryo which could be extremely serious. A PGD misdiagnosis for DM leading to the termination of an affected pregnancy has previously been reported (Sermon, 1998). If a highly heterozygous STR is co-amplified with the DM repeat, the possibility that both repeats from unrelated DNA would amplify the same profile as that expected from the parent, is greatly reduced. Even if one or both parents are homozygous for the STR, the detection of unexpected STR profiles would be recognized as contamination and therefore the embryo would not be transferred. It could also reduce the chances of misdiagnosis due to amplification of DNA from a related source such as cumulus cells surrounding the oocyte or spermatozoa attached to the zona pellucida. As long as the parents are heterozygous for the STR, related DNA should amplify both copies of the STR. This would be recognized as contamination when analysing the results in the embryos, as a blastomere should only show amplification of one copy of the STR from each parent. To minimize the risk of related DNA contamination from either parent, it is imperative to ensure that all cumulus is stripped from the oocytes and that ICSI is performed when PGD is undertaken for a single gene defect.
In our experiments, the amplified PCR products from single lymphocytes, pre-clinical and clinical blastomeres, matched the expected genotype. For single lymphocytes the profiles were identical to the genotypes of the donors (Table II), while in the pre-clinical blastomeres they corresponded to the profiles expected from the parental genotypes. The same was observed on the day of clinical PGD where there was no amplification of an unexpected fragment size for either DM or the D195219 STR in any of the blastomeres, thereby indicating that the results obtained were the true genotypes of the embryos and DNA contamination was unlikely.
Second, multiplex fluorescent PCR protocols can reduce and aid detection of ADO. ADO is a major concern of single cell PCR and is the failure of amplification of one allele from a known heterozygous cell (ADO in homozygous cells cannot be detected). Although no single protocol has been able to eliminate ADO completely, PCR sensitivity can be increased 1000-fold by the use of fluorescent PCR (Hattori et al., 1992) and this increased detection of PCR products can reduce ADO. Findlay and colleagues were the first to demonstrate the advantages of fluorescent PCR over conventional PCR for reducing ADO (Findlay et al., 1995). They concluded that a number of cases of ADO are in fact preferential amplification of one of the alleles and that the other weakly amplified product is not detected by conventional analysis. Due to the increased sensitivity of fragment analysis, cases of true ADO and extreme preferential amplification can be distinguished, as long as the x-axis of any fluorescent PCR print-out is set to produce flat background lines, thereby removing unspecific background peaks. We were able to keep our background lines flat (Figure 1) and noted preferential amplification of one of the alleles in single cells from different genotypes (for example see Figure 1b, donor 4), which was not related to the size of the repeat unit. In the samples where true ADO occurred, there was random failure of amplification of one allele or the other which also was not associated with the size of the repeat.
Another way of detecting ADO, after linkage analysis in an informative family, would be by incorporating a closely linked marker, such as an STR, in the PCR. Multiplex PCR, with the inclusion of a linked polymorphic marker, have been used in other single gene defects to act as an internal control for ADO (Wu et al., 1993; Ao et al., 1998; Rechitsky et al., 1998). During the review of this manuscript, a paper was published describing a single round fluorescent PCR for DM using a chromosome 19 STR in conjunction with a chromosome 21 STR (Piyamongkol et al., 2001) with 40 cycles being required to visualize the product. We found two rounds of PCR assay, with a total of 40 cycles, to be more reliable than a single round of PCR (unpublished data). The ability to adjust the PCR conditions in the second round for different loci independently provided the flexibility to develop the assay with greater sensitivity. In our clinical PGD case, we did not perform linkage analysis in the family of the affected patient, because the STR that was informative for PGD was uninformative for linkage analysis (i.e. the affected patient was homozygous for the STR). Therefore, there was a possibility that an embryo diagnosed as affected (i.e. amplifying only the male allele and a full STR profile) in fact had ADO of the normal female repeat. The ADO rate on the day of the clinical PGD for DM given as 0% could have been inaccurate if based only on the results of that day. However, this conclusion was not reached merely according to the clinical PGD but in conjunction with the diagnoses obtained with blastomeres tested after the PGD. Every embryo that had been diagnosed as affected on the day of PGD showed the same profile in the remaining blastomeres.
Even though the STRs were not useful for linkage analysis in our clinical case, because they are very closely mapped to the DMPK region of chromosome 19 (with 0% and 2% recombination frequency), they could be useful for other informative couples. Addition of a linked marker would allow a differentiation between failure of amplification (ADO) of the healthy DM allele from the affected parent and an affected embryo (i.e. presence of only the expanded allele of the affected parent). Clinically of more importance, diagnosis could be made based not only on the detection of a normal allele from the affected individual but also the amplification of the STR profile corresponding to that carried on the normal allele. This would further reduce the chances of failure to detect an affected embryo.
The STR, in our clinical PGD, served to identify another complication of single cell PCR, namely, chromosomal mosaicism. In one of the embryos, both biopsied blastomeres amplified only the affected parent's normal DM repeat. If DM alone had been amplified, this embryo would be diagnosed as having failure to amplify the male allele, i.e. ADO. Due to the inclusion of the STR profile that also only amplified the female allele, we were able fairly confidently to diagnose this embryo, at the time of PGD, as monosomic or mosaic for at least chromosome 19. Therefore, on the day of PGD, we did not report ADO for this embryo, a conclusion verified from further blastomeres of this embryo that showed the same profile.
Third, additional genetic information gained from inclusion of STR profiles in clinical PGD can maximize the number of embryos in which a genotype can be assigned. The more loci amplified, the greater the chances of identifying normal embryos. If two blastomeres from one embryo are tested, it could be that one shows normal DM alleles from both parents but the second cell shows only the normal allele from the affected patient. In such a scenario, if both cells amplified a full STR profile, we could stipulate the result as ADO of the DM allele from the unaffected parent, i.e. a normal embryo. Without the STR we would not be able to say if the second blastomere had ADO or was chromosomally abnormal. Maximizing the number of diagnosed embryos is particularly important in the case of dominant diseases because only 50% of all embryos generated will be expected to be normal and therefore available for transfer. The diagnosis of a maximum number of embryos allows a greater selection of the best unaffected embryos for transfer, thus providing the highest chance of a successful pregnancy.
Another way to reduce the possibility of misdiagnosis is to obtain results from two blastomeres of an embryo (Ao et al., 1998; Ray et al., 1998). We generally biopsy two cells for PGD of single gene defects but unfortunately, in this instance, only three of the nine embryos had 7 cells on day 3. The removal of two blastomeres in an embryo with 
6 cells is believed to be detrimental to future embryo development (Krzyminska et al., 1990; Tarin et al., 1992), therefore the diagnosis for six embryos was performed only on a single blastomere. An embryo that does not reach the 7-cell stage by 72 h of development is less likely to progress to the expanded blastocyst stage (8.1% for 6 cells compared to 43.4% for 7 cells) (Shapiro et al., 2000). Therefore the six slow-growing embryos in our clinical PGD were less likely to implant. Of the three embryos where two blastomeres were tested, one was diagnosed as normal in both cells, one affected in both cells, and the third amplified only the female alleles for both DM and the STR. Of the six embryos where one blastomere was tested, two were diagnosed as normal, three affected and one failed to amplify the DM alleles. At embryo transfer, two embryos diagnosed as normal with full DM profiles were transferred. Any non-transferred embryos had further blastomeres genotyped and, in all cases, the genotype confirmed that made on the day of PGD.
Ideally, if both expanded and normal DM alleles can be amplified reliably at the single cell level, direct diagnosis of an affected embryo will be possible. In the absence of this, our fluorescent multiplex PGD permits an accurate diagnosis for DM and is applicable to many DM-affected couples.
Acknowledgements
We would like to thank the staff of the McGill Reproductive Centre for helping in the management of the clinical PGD case reported in this study.
Notes
4 To whom correspondence should be addressed. E-mail: asangla.ao{at}muhc.mcgill.ca 
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Submitted on March 12, 2001; accepted on July 9, 2001.
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