Molecular Human Reproduction, Vol. 9, No. 9, 559-567,
September 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
Improving clinical preimplantation genetic diagnosis for cystic fibrosis by duplex PCR using two polymorphic markers or one polymorphic marker in combination with the detection of the 
F508 mutation
Submitted on February 14, 2003; resubmitted on April 24, 2003. accepted on May 2, 2003
1 Centre for Medical Genetics and 2 Centre for Reproductive Medicine, University Hospital and Medical School, Dutch-speaking Brussels Free University, Laarbeeklaan 101, 1090 Brussels, Belgium
3 To whom correspondence should be addressed. e-mail: veerle.goossens{at}az.vub.ac.be
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ABSTRACT |
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Cystic fibrosis (CF) is an autosomal recessive disease characterized by obstruction and chronic infection of the respiratory tract and pancreatic insufficiency. The first preimplantation genetic diagnosis (PGD) for CF was carried out in 1992. At our centre the first cycle was performed in 1993. However, the number of known CF mutations is >1000, so developing mutation-specific PCR protocols for PGD is unfeasible. This is why a number of marker-based duplex PCRs were developed at the single cell level. A duplex PCR of a mutation and one or two microsatellites is not only a diagnostic tool, but it can also be used as a control for allele drop-out and contamination. During PGD, embryos obtained in vitro are analysed for the presence or absence of a particular genetic disease, after which only embryos shown to be free of this disease are returned to the mother. In total, 22 PGD cycles with duplex PCR (IVS8CA/IVS17BTA,
F508/IVS8CA, F508/IVS17BTA and D7S486/D7S490) were carried out in 16 couples, which resulted in four ongoing pregnancies and one miscarriage. Key words: cystic fibrosis/duplex PCR/preimplantation genetic diagnosis
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Introduction |
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Cystic fibrosis (CF) is one of the most common severe, lethal genetic disorders (Zielenski and Tsui, 1995; Welsh, 1995), and the first non-X-linked monogenic disorder for which preimplantation genetic diagnosis (PGD) was performed (Handyside et al., 1992). The median life expectancy is now >30 years and it is projected that in newborn infants it will become >40 years. But although the life expectancy has improved beyond all recognition, the quality of life is far from good (Doull, 2001). The carrier frequency in the Caucasian population of this autosomal recessive disorder is 1 in 25; 1 in 2500 newborns is affected (Welsh, 1995). Clinical characteristics are obstruction and chronic infections of the respiratory tract and pancreatic insufficiency. The cystic fibrosis transmembrane conductance regulator (CFTR) gene, which causes CF when mutated, is located on chromosome 7q22-31 (Riordan et al., 1989). A phenylalanine deletion at amino acid position 508 is the most common mutation and accounts for
70% of the CF alleles worldwide. The remaining known CF alleles carry other mutations (>1000 listed, www.genet.sickkids.on.ca/cftr), of which some occur with a frequency of a few per cent, although most are private mutations (occurring only once in the whole population). Congenital bilateral absence of the vas deferens (CBAVD) is a particular form of CF, which leads to male infertility due to obstructive azoospermia. Sperm can be retrieved by fine needle aspiration (FNA) and used for IVF through ICSI (Van Steirteghem, 1997). CBAVD patients are assumed to be compound heterozygotes for two different CFTR mutations or to have one CFTR mutation in combination with a 5T ‘splice’ variant in intron 8 (Lissens et al., 1996).
PGD can be considered as an early form of prenatal diagnosis which circumvents the problem of therapeutic abortion. The technique has become possible due to the simultaneous development of IVF, micromanipulation of the embryo, the PCR for monogenic disorders, and fluorescence in-situ hybridization (FISH) for chromosomal abnormalities. With PGD, only embryos diagnosed at the preimplantation stage as being unaffected by the disease under consideration are transferred to the uterus. Since genetic analysis is performed on one or two single blastomeres, it has to meet high standards of efficiency, including low allele drop-out (ADO) rates and contamination control (Lissens and Sermon, 1997). We have previously described a number of marker-based protocols for CF, because mutation-specific PCR protocols for every possible CF mutation are not feasible (Goossens et al., 2000). For this purpose we used three intragenic polymorphic markers IVS8CA, IVS17BTA and IVS17BCA, and four extragenic polymorphic markers, D7S490, D7S486, D7S480 and D7S523. IVS8CA is a CA-repeat in intron 8 of the CFTR gene with 12–24 repeats and a heterozygosity of 48%. IVS17BCA is a CA-repeat in intron 17B with 11–20 repeats and 39% heterozygosity. The IVS17BTA marker is a TA-repeat in intron 17B and has 7–54 repeats with 87% heterozygosity (Zielenski et al., 1991). The extragenic polymorphic markers are also CA-repeats with heterozygosity rates of 80, 77, 87 and 81% respectively (Dreesen et al., 2000).
Here we describe duplex PCRs that amplify two markers or one marker combined with the F508 mutation.
This approach allows us to offer PGD to couples in whom the mutation(s) is (are) unknown or occur(s) with low frequency. Moreover, a duplex PCR for these markers has to be developed once and can then be applied to all couples who are informative. Informativity means that chromosomes carrying the mutation can be identified. By analysing family members, it is possible to determine which alleles co-segregate with CF. If the mutations are not known, the couple must have an affected child in order to be able to analyse which alleles co-segregate with the mutation.
A duplex PCR of a mutation and a linked marker is not only a diagnostic tool, but it can also be used as a control for ADO and contamination. For example, if, during a PGD, two marker alleles, coupled to the wild-type CF allele of both partners, are observed in an embryo, then only one fragment representing the wild-type allele should be observed. The finding of a carrier genotype with mutant and normal CF alleles would indicate contamination in the PCR carried out for the mutation or recombination between the marker and the mutation. If, on the other hand, analysis of the marker alleles indicates one allele linked to the wild-type allele and one linked to the mutant CF allele, a carrier CF genotype is expected. If, however, only a wild-type CF allele is observed, the conclusion of ADO can be drawn. Without co-amplification of the marker, these embryos could be diagnosed incorrectly, which could lead to the transfer of affected embryos. Undetected ADO of the normal CF allele on the other hand leads to the loss of unaffected embryos (Dreesen et al., 2000; Eftedal et al., 2001; Moutou et al., 2002).
Here we present the development and clinical application of four duplex PCR: a PCR for the marker IVS8CA in combination with IVS17BTA, one for the F508 mutation and the marker IVS8CA, a PCR for the duplex F508 and IVS17BTA and a fourth duplex PCR of D7S486 and D7S490.
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Materials and methods |
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Collection of single lymphoblasts
Lymphoblasts transformed by the Epstein–Barr virus (EBV) were cultured as described by Ventura et al. (1988). A few colonies were collected and washed three times in 500 µl phosphate-buffered saline (PBS).
Single lymphoblasts were then collected as described earlier by Sermon et al. (1998). Briefly, they were transferred to droplets of Ca2+- and Mg2+-free medium, washed in three different droplets and transferred blindly to a PCR tube containing 2.5 µl alkaline lysis buffer (ALB; 50 mmol/l dithiothreitol, 200 mmol/l KOH or 200 mmol/l NaOH) (Li et al., 1988). An aliquot from the last washing droplet was taken to serve as a blank.
Patient work-up
Ethical approval was granted for the study from the institutional ethical committee and all patients gave informed consent. Couples requesting PGD for CF in whom two or more (as in CBAVD and CF patients) different mutations were found or in whom the mutation was not yet known were checked for informativity. A fluorescent PCR, using the polymorphic markers, was therefore carried out on the couples’ genomic DNA, extracted from peripheral blood. A couple was considered to be informative if the affected alleles from both partners could be clearly distinguished from the non-affected. This meant that both partners of the couple were heterozygotes for one of the polymorphic markers and that segregation from the affected alleles could be determined. It is not necessary for all polymorphic alleles to be different.
Table I shows the different mutations that were encountered as well as relevant clinical information on the 32 couples checked for informativity of CFTR markers.
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ICSI procedure
ICSI was performed in all cycles to prevent contamination with sperm adhering to the zona pellucida (ZP) and to ensure fertilization (Liebaers et al., 1998). The ICSI procedure was carried out as described by Joris et al. (1998).
Briefly, the patients underwent controlled ovarian stimulation by administration of a GnRH agonist or antagonist in association with human urinary menopausal or recombinant gonadotrophins and hCG (Vandervorst et al., 1998).
Oocyte retrieval was carried out by vaginal ultrasound-guided puncture of the ovarian follicles, 36 h after hCG was administered. The cells of the cumulus oophorus and corona radiata were removed (Van de Velde et al., 1997) and metaphase II oocytes were injected with a single sperm into the ooplasm. Ejaculated sperm or testicular sperm after fine needle aspiration were used (Van Steirteghem, 1997). The injected oocytes were incubated at 37°C in 25 µl of G1/G2 medium in an incubator (37°C, 5% O2, 5% CO2, 90% N2) (Heraeus; Vander Heyden, Belgium). After 16–18 h, fertilization was assessed and further development of the embryos was evaluated on days 2 and 3 prior to biopsy.
Embryo biopsy
A hole was made in the ZP using a non-contact 1.48 µm diode laser system (Fertilase; MTM Medical Technologies, Switzerland) coupled to an inverted microscope in order to deliver two or three laser pulses of 16 ms to the ZP, creating a funnel-shaped hole (Sermon et al., 1999).
One or two blastomeres were then gently aspirated through the hole. Embryos in which compaction had already started were incubated for 5–10 min in Ca2+- and Mg2+-free medium (In Vitro Life, Sweden) before biopsy.
Further procedures were then performed using a stereomicroscope. Each blastomere was washed three times in (home-made) Ca2+- and Mg2+-free medium (Sermon et al., 1998) and transferred to a 0.2 ml PCR tube containing 2.5 µl ALB. They were kept at –80°C for 
30 min (Sermon et al., 1998).
Cell lysis
The cells in ALB were further lysed by incubating them at 65°C for 10 min (Sermon et al., 1995). After lysis the PCR tubes were immediately put on ice.
The ALB was neutralized afterwards with 2.5 µl neutralization buffer (NB) (0.9 mol/l Tris–HCl; pH 8.3, 0.3 mol/l KCl, 0.2 mol/l HCl) for KOH-containing ALB or with 2.5 µl Tricine (500 mmol/l; pH 4.9) for NaOH-containing ALB (Sermon et al., 1998).
PCR procedures
The procedures used to perform the intragenic and extragenic marker-based duplex PCR are summarized in Table II. Two consecutive PCR rounds were performed in all four duplex PCR. Three microlitres from the first PCR round were taken as a template for the second round PCR (Goossens et al., 2000). The reason for separating our PCR reactions after 10 cycles is that we started from published primer sequences without any adaptations with regard to annealing temperature or fragment length. Some of the fragments did overlap in length, which meant that we were not able to detect them by using the ALF, since only one fluorescent label is available. In addition we also noticed that the efficiency of the PCR is often much better when the reaction is separated. Primer sequences are given in Table III. The primers were labelled with Indocarbocyanine (CY5) since electrophoresis was performed on an automated laser fluorescence DNA sequencer (Amersham Pharmacia Biotech, The Netherlands).
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Results |
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Informativity
In a first stage, the informativity of all candidate couples was checked for the three intragenic polymorphic markers IVS8CA, IVS17BTA and IVS17BCA and/or for the four extragenic polymorphic markers D7S486, D7S480, D7S490 and D7S523. Therefore, PCR was carried out on 100 ng of the couples’ genomic DNA as well as on the DNA of affected children if present or of the patients’ parents. Table IV shows the results of the informativity testing.
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In total, informativity was analysed in 32 couples. Twenty couples were only tested for the three intragenic polymorphic markers. Seven couples were found to be informative for both IVS17BTA and IVS8CA. Nine other couples were informative for the IVS17BTA polymorphism only while two were informative for IVS8CA. Two couples remained uninformative (not shown in Table IV). Twelve out of the 32 couples were also checked for the extragenic polymorphic markers. One couple was informative for all three intragenic markers but for none of the extragenic markers. Two couples were informative for D7S480 only, two other couples were informative for D7S486 and D7S490. One couple was found to be informative for D7S480, D7S490 and D7S523. One other couple was informative for the three extragenic markers: D7S480, D7S486 and D7S490. One couple was informative for all extragenic markers D7S480, D7S486, D7S490 and D7S523. Furthermore, two couples were informative for IVS17BTA and D7S480, D7S486 and D7S490. One couple was informative for IVS17BCA, D7S480, D7S486, D7S490 and D7S523. For one couple, diagnosis remained impossible using this strategy, since they were uninformative for all seven markers (not shown in Table IV). If one takes into account the patients who were checked for the intragenic markers only and the patients checked for all seven markers, 29 out of 32 couples were informative for at least one polymorphic marker.
Lymphoblasts from heterozygous individuals
To develop a single cell PCR procedure, lymphoblasts from heterozygous individuals were analysed to evaluate the amplification, the allele drop-out (ADO) and the contamination rate.
The results are summarized in Table V. The amplification rate for all four duplex PCR ranges from 92 to 98%, after split of the reaction. The ADO rate in the four PCR ranges from 0 to 4.6%, while 0–4.4% of the blanks were contaminated.
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Data from simplex PCR that were developed earlier (Goossens et al., 2000) have been included for comparison of amplification, ADO and contamination rates.
Clinical PGD procedures
Eighteen of the informative couples have already undergone PGD in 31 cycles. Twenty-two cycles in which a biopsy could be performed (16 couples) were carried out with a duplex PCR: two cycles with the duplex IVS8CA/IVS17BTA, two cycles with the duplex F508/IVS8CA, fifteen cycles with the duplex F508/IVS17BTA and three cycles with the duplex D7S486/D7S490. Two started cycles (one with the duplex F508/IVS17BTA and one with the duplex D7S486/D7S490) remained without biopsy because no good quality embryos were available.
In total, 253 cumulus–oocyte complexes were obtained and ICSI was performed in 211 metaphase II oocytes (83.4%). A total of 164 injected oocytes (77.7%) showed two pronuclei (2PN), proving normal fertilisation. Of these, 108 (65.8%) cleaved further beyond the 6-cell stage and could be biopsied removing one or two blastomeres. Because of a recently started study that compares the removal of one versus two blastomeres, only one blastomere was removed in four cycles. Thirty-six of all biopsied embryos (33.3%) were affected, 46 embryos were carriers (42.6%) and nine (8.3%) were unaffected. Seventeen embryos remained without diagnosis (15.7%). Forty-two embryos were transferred in 21 transfers (2.05 embryos per transfer). Total pregnancy rates were 23.8% per transfer, 22.7% per PGD cycle with biopsy and 20.8% per started PGD cycle (Table VI).
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In the first two cycles (two couples) with the duplex IVS8CA/IVS17BTA, six embryos were analysed, two were carriers, three were affected and one remained without diagnosis. The two carrier embryos were transferred, one in each cycle, but the patients did not achieve pregnancy.
The two cycles (one couple) with the duplex F508/IVS8CA gave the following results. Seven embryos were analysed, five were carriers, one was affected and one was unaffected. In the first cycle, two carrier embryos were transferred. The second transfer cycle with two carrier and one healthy embryo resulted in an ongoing pregnancy.
In the 15 cycles to biopsy (10 couples) with the duplex F508/IVS17BTA, 80 embryos were analysed, of which seven were unaffected, 33 were carriers and 29 were affected. Eleven embryos remained without diagnosis. In total, 30 embryos were transferred in 14 transfer cycles. One cycle ended without transfer due to technical problems: the dish with embryos was mixed up in the IVF laboratory and it was impossible to identify the healthy embryos afterwards. In the other cycles three pregnancies occurred of which one ended in a miscarriage. One twin pregnancy is ongoing. The other pregnancy resulted in the birth of a healthy boy.
There were three cycles to biopsy with the duplex D7S486/D7S490 in three couples. Fifteen embryos were analysed. Six embryos were carriers, three were affected, one embryo was unaffected and five remained without diagnosis. Five embryos were transferred in three transfer cycles. One patient became pregnant and delivered a healthy boy.
When no diagnosis was obtained (in total: 17 out of 108 embryos i.e. 15.7%), it was a result of either ADO in one or two cells or total failure of cell amplification. In this series of blastomeres no discordant results were obtained. Diagnosis is of course only assigned when both cells yield the same result, or when both loci provide concordant results if only one cell result is available (one cell biopsy or amplification failure in one cell). Amplification efficiency and ADO are shown in Table VII.
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Discussion |
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TopABSTRACTIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Here we have described the development and clinical application of preimplantation genetic diagnosis for CF using four duplex PCR. In total, 16 couples were treated. In a preclinical setting, the single cell duplex PCR were tested on single lymphoblasts.
Until now we have used the ALF system for fragment analysis. To avoid overlapping fragments while using this single colour system, we would like to switch to a multicolour system such as the ABI prism 3100-Avant Genetic Analyzer (Applied Biosystems). This would make it possible to detect two fragments of the same length.
The use of polymorphisms in duplex PCR (either two different polymorphisms or one polymorphism combined with the F508 detection) can be important on three levels.
Initially, we used polymorphisms when the partners carried different mutations. The diagnosis of compound heterozygote embryos using a duplex for two different mutations can lead to the transfer of affected embryos if ADO of the affected allele occurs in one mutation. This is why in the past we used to analyse only one mutation in two cells and transferred only embryos free from the mutation (Goossens et al., 2000). Carrier embryos for the analysed mutation cannot be transferred through this approach because of their unknown status for the second mutation. However, half of these embryos are indeed free of the second mutation, which means that healthy carrier embryos are lost for transfer. By means of polymorphisms, two or more (CBAVD, CF patients) different mutations can be indirectly analysed at once, decreasing the possible loss of healthy embryos for transfer.
A second advantage of these polymorphisms in duplex with a second polymorphism or a specific mutation detection is that ADO and contamination, two well-known problems of single cell PCR, can be detected.
A very important third reason is that so far >1000 CF mutations have been identified. It would be very labour-intensive to develop a PCR assay for all PGD patients presenting different mutations. It is much easier to develop a battery of tests for polymorphisms to be used instead of direct mutation analysis. By using four extragenic polymorphic markers, two on both sides of the gene, and three intragenic polymorphic markers, the whole gene can be covered without too large a risk for recombination (2.2–6% between the most 3' and the most 5' marker) (Dreesen et al., 2000). It must be stressed that if the markers are well chosen in relation to the mutations, recombination will lead to the embryos not being assigned a diagnosis, rather than to transfer of affected embryos.
We would like to emphasize that although the heterozygosity rates of IVS17BTA (87%), IVS8CA (48%), IVS17BCA (39%), D7S523 (81%), D7S486 (77%), D7S480 (87%) and D7S490 (80%) are relatively high and the F508 mutation accounts for 70% of all CF mutations, this does not mean that the couples are also informative. This is often the case in families where there are no affected children and the informativity testing has to rely on the patient-couple and their respective parents. We often see individuals who are heterozygotes, but the same combination of allele sizes is often observed in a family. This leads to a high heterozygosity rate, but a lower informativity rate.
In our 32 patient-couples, there were 27 couples (84.37%) in whom at least one of both partners carried a F508 mutation. In thirteen of the 27 couples (48%) for whom a duplex PCR was developed, the duplex IVS17BTA/F508 could be used. In three out of 27 (11.11%) cases IVS8CA/IVS17BTA could be used and in two out of 27 (7.41%) cases IVS8CA/F508 was used. For the extragenic polymorphic markers in four out of 27 (14.81%) patients, D7S486/D7S490 was used, and for the combinations D7S480/D7S523 and D7S480/M470V (no PGD cycles yet) two out of 27 (7.41%) couples were informative each time. In total, six duplex PCR were developed, useful for 27 couples out of the 32 couples checked for informativity (84.37%). Moutou et al. (2002) presume that by using the combinations IVS17BCA/F508 and IVS8CA/F508, 80% of their patient- couples can be helped. Our own numbers are therefore much lower than would be expected in theory. This means that a whole battery of duplex PCR will have to be developed. A more efficient solution would be to develop a multiplex of all extragenic/intragenic markers in combination with the F508 mutation (Dreesen et al., 2000).
So far, five pregnancies in four patients have resulted from our 22 PGD with duplex PCR for CF. One patient had a miscarriage but became pregnant a second time. In two patients the PGD results were confirmed by prenatal diagnosis. The two other patients refused prenatal diagnosis because of objections against abortion. Two healthy babies were born.
The amplification and ADO rates obtained during PGD did not differ from the preclinical data obtained with lymphoblast analysis, showing the usefulness of these cells as a model. The relatively low number of homozygous normal embryos (8.3%) is partially due to the presence of two CBAVD patients and two CF patients, who cannot have homozygous normal embryos. If we exclude these patients, there are eight homozygous normal embryos (11.5%), which is still somewhat lower than expected.
Development of duplex PCR with other combinations of markers and the development of multiplex PCR will continue in order to be able to help almost all couples requesting PGD for CF.
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Acknowledgements |
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The authors wish to thank the clinical, paramedical and laboratory staff at the Centres for Medical Genetics and Reproductive Medicine for their help and commitment. Furthermore we are grateful to J.Deconinck of the Language Education Centre at our University. This work was supported by grants from the Belgian Fund for Scientific Research, Flanders (FWO-Vlaanderen). V.G. was funded by the Forton Foundation and is now supported by grants from Fund for Scientific Research Flanders. K.S. and and M.D.R. were supported by grants from the Funds for Scientific Research Flanders. B.S. is employed by the Forton Foundation.
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G. Altarescu, B. Brooks, Y. Kaplan, T. Eldar-Geva, E.J. Margalioth, E. Levy-Lahad, and P. Renbaum Single-sperm analysis for haplotype construction of de-novo paternal mutations: application to PGD for neurofibromatosis type 1 Hum. Reprod., August 1, 2006; 21(8): 2047 - 2051. [Abstract] [Full Text] [PDF]
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J. F. Sanchez-Garcia, J. Benet, C. Gutierrez-Mateo, J. Luis Seculi, E. Monros, and J. Navarro Multiple mutation analysis of the cystic fibrosis gene in single cells Mol. Hum. Reprod., June 1, 2005; 11(6): 463 - 468. [Abstract] [Full Text] [PDF]
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C. Spits, M. De Rycke, N. Van Ranst, H. Joris, W. Verpoest, W. Lissens, P. Devroey, A. Van Steirteghem, I. Liebaers, and K. Sermon Preimplantation genetic diagnosis for neurofibromatosis type 1 Mol. Hum. Reprod., May 1, 2005; 11(5): 381 - 387. [Abstract] [Full Text] [PDF]
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