Mol. Hum. Reprod. Advance Access originally published online on April 15, 2005
Molecular Human Reproduction 2005 11(5):381-387; doi:10.1093/molehr/gah170
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Preimplantation genetic diagnosis for neurofibromatosis type 1
Research Centre Reproduction and Genetics, University Hospital and Medical School, Dutch-speaking Brussels Free University, Laarbeeklaan 101, 1090 Brussels, Belgium
1 To whom correspondence should be addressed. Email: martine.derycke{at}az.vub.ac.be
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Abstract |
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PGD is an alternative to prenatal diagnosis that circumvents therapeutic abortion. Diagnosis is carried out on single cells obtained from three-day-old embryos, and only those that are free of the disease under consideration are transferred to the mother. Neurofibromatosis type 1 (NF1) is a common neurocutaneous disorder, inherited as an autosomal dominant trait and caused by mutations in the NF1 gene. For some patients, PGD may be the only acceptable manner to ensure the birth of unaffected children. Because of the large number of known NF1 mutations, the development of mutation-specific single-cell protocols is impractical, labour-intensive and expensive. This paper discusses the development of five PGD protocols, three of which are based on multiplex PCR for microsatellite-markers linked to the NF1 gene. After a linkage study, the diagnosis can be established through the markers, thereby obviating the need to detect the mutation itself. This not only ensures the accurate diagnosis of the embryos, but also a prompt acceptance of PGD referrals since one protocol can be useful for several couples. In addition, two mutation-specific PCRs were developed for two couples where a marker-based protocol was not applicable. In total, 16 PGD cycles were carried out for six couples, which resulted in one ongoing pregnancy and the delivery of a healthy unaffected boy.
Key words: neurofibromatosis type 1/NF1/PGD/single-cell PCR
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Introduction |
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Neurofibromatosis type 1 (NF1; OMIM# 162200 [OMIM] ) is one of the most frequent Mendelian disorders, with a prevalence of 1 in 3500 live births. It is a neurocutaneous disorder characterized by autosomal dominant inheritance with complete penetrance and a very variable expression (Friedman, 1999
). The main clinical symptoms, on which the diagnosis is based, are multiple café-au-lait spots, (sub)cutaneous neurofibromas, plexiform neuromas, axillary or inguinal freckling, optic gliomas and iris Lisch nodules. Since 10–20% of the neurofibromas develop into malign peripheral nerve sheath tumours, NF1 can be considered as a cancer predisposition syndrome (Upadhyaya et al., 2004). The commonest neurological complication of NF1 is cognitive impairment during childhood, which is often the major concern of parents of affected children (North, 2000).
The NF1 transcript structure spans 280 kb on chromosome 17q11.2, and is generally classified as a tumour suppressor gene. It consists of 58 exons that encode the neurofibromin protein. Additionally, the sequence of NF1 contains the gene OMGP and the viral insertion sites EVI2A and EV12B in intron 36–37 and the pseudogene AK3P1 in intron 48–49 (Ensembl, The Wellcome Trust Sanger Institute, http://www.ensembl.org, Ensembl ID: ENSG00000196712). The mutation rate in the NF1 gene is one of the highest reported in any human disorder, leading to approximately 50% of sporadic cases (Friedman, 1999). Currently, over 500 different NF1 mutations have been described without any clearly predominant mutation (Human Gene Mutation Database, http://archive.uwcm.ac.uk/uwcm/mg/hgmd0).
PGD is an alternative to prenatal diagnosis in which the genetic diagnosis is performed on single blastomeres obtained from 3-day-old IVF embryos (Sermon et al., 2004). Only embryos diagnosed as free of the disorder under consideration are transferred to the mother, so that therapeutic abortion is avoided. PGD often appears as an ethically acceptable way of avoiding less generally accepted indications for pregnancy termination such as cancer predisposition syndromes or late onset diseases (Robertson, 2003). This is why, for many NF1 patients, PGD is a suitable option for avoiding the birth of an affected child.
The development of an efficient and reliable single-cell PCR-based assay for PGD is labour-intensive and expensive. In the case of disorders for which a large number of private mutations have been described, developing mutation-specific protocols is impractical and highly inefficient. A more practical approach is the development of a multiplex PCR for markers linked to the gene. This indirect diagnosis makes it possible to apply the same PGD protocol to different couples, irrespective of their mutation. Furthermore, the analysis of multiple genotypes in one sample increases the reliability of PGD (Dreesen et al., 2000; De Vos et al., 2003; Goossens et al., 2003). The large number of NF1 mutations and the high demand for PGD for the disorder make it a good candidate for this strategy.
The aim of this work was to develop NF1 PGD protocols in response to the current requests and to use when possible a linked marker approach in order to improve the reliability of PGD. This enables us to respond more efficiently to future PGD referrals.
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Materials and methods |
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Patient description
The relevant details of the six couples that underwent PGD for NF1 are summarized in Table I. Approval for the present study was obtained from the institutional committee of medical ethics (University Hospital and Medical School, Faculty of Medicine and Pharmacy, Dutch-speaking Brussels Free University) and all patients gave informed consent.
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Collection and lysis of single cells
Epstein–Barr virus-transformed lymphoblasts, cultured as described elsewhere (Ventura et al., 1988
), were used as a model to develop single-cell PCRs. Single lymphoblasts were collected in alkaline lysis buffer (50 mM dithiothreitol, 200 mM NaOH; Li et al., 1988) as described elsewhere (Goossens et al., 2003), and further lysed by incubating at 65°C for 10 min. Single-sperm cells were collected to perform segregation studies as described elsewhere (De Vos et al., 2003) and treated likewise lymphoblasts.
ICSI procedure
The patients underwent controlled ovarian stimulation with GnRH antagonist and recombinant follicle-stimulating hormone (Platteau et al., 2002). Transvaginal ultrasound-guided oocyte retrieval was done 36 h after HCG administration.
ICSI was used in all cycles instead of IVF to prevent contamination of the PGD with residual sperm adhered to the zona pellucida (Liebaers et al., 1998). The ICSI procedure on mature metaphase-II oocytes was performed as described by Devroey and Van Steirteghem (2004). Fertilization was assessed after 16–18 h and embryo development was further evaluated on day 2 and 3 prior to biopsy.
Embryo biopsy
The embryo biopsy was performed on the morning of day 3 post-fertilization (Joris et al., 2003). Only embryos at a 6-cell or later stage of development were biopsied. A hole was made in the zona pellucida using two or three laser pulses of 5–7 ms of a non-contact 1.48 µm diode laser system (Fertilase; Octax, Herbron, Germany) coupled to a micromanipulator on an inverted microscope. One, two, or exceptionally, three blastomeres containing a nucleus were gently aspirated through the opening. The single blastomeres were collected and further treated like the single lymphoblasts.
Microsatellite polymorphic markers and PCR procedures
Two intragenic and two extragenic microsatellite polymorphic markers were used to develop the different protocols. Primers and location of IVS27AC28.4 (intron 27) and IVS38GT53.0 (intron 38) were obtained from their first publication (Lazaro et al., 1993, 1994). According to the nucleotide database (NCBI, http://www.ncbi.nlm.nih.gov), D17S841 is located approximately 1.9 Mb upstream and D17S1800 0.2 Mb downstream of the NF1 gene. Primer sequences were obtained from NCBI and Lopez Correa et al. (1999). The primers for the c.2975T > G and c.1529C > T mutations were adapted from Li et al. (1995). The primer sequences are summarized in Table II.
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Fluorescent PCRs were performed on 100 ng of genomic DNA to test the informativity of the couples for the different polymorphic markers and the segregation patterns of their mutation with the markers. The PCR protocol used was the same for all markers. The reaction mix contained 200 µmol/l of each dNTP (Amersham Pharmacia Biotech, Roosendaal, The Netherlands), 0.4 µmol/l of both forward and reverse primers (Eurogentec, Seraing, Belgium), 1 x Expand High Fidelity (EHF) buffer 2 and 0.875 IU EHF polymerase (EHF system, Roche, Brussels, Belgium) in a final volume of 25 µl. The PCR programme was as follows: 5 min denaturation at 95°C, followed by 30 cycles of 30 s at 95°C, 30 s at 60°C and 30 s at 72°C and a final extension of 5 min at 72°C. The samples were analysed on the automated laser fluorescence DNA sequencer ALFExpress II, using the Allelelinks software provided by the manufacturer (Amersham Pharmacia Biotech) or the ABI PRISM 3100—Avant Genetic analyser (Applied Biosystems, Nieuwerkerk a/d Ijsel, The Netherlands).
Four multiplex single-cell PCRs for microsatellite markers and two duplex single-cell PCRs for a mutation and a marker were developed for PGD; the PCR protocols are summarized in Table III. Markers D17S841 and D17S1800 are co-amplified with either IVS27AC28.4 or with IVS38GT53.0 in two different triplex PCRs, using primers labelled for the ALFExpress II (with CY5). The triplex PCR for D17S841, D17S1800 and IVS38GT53.0 was later adapted for analysis on the ABI PRISM 3100—Avant Genetic analyser. The fourth protocol involved the co-amplification of markers IVS27AC28.4 and IVS38GT53.0 in a duplex PCR. Two mutation-specific protocols were developed, one in which the mutation c.1529C > T was combined with IVS38GT53.0, and another which combined the c.2975T > G mutation with IVS27AC28.4.
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All the triplex PCRs for microsatellite markers were performed in a two-rounds system. After 10 cycles of coamplification of the three primer sets together (first round), the reaction was stopped and two new PCRs were set up in which only one or two markers were amplified per reaction, using 3 µl of the first round as a template (second round).
For the detection of the c.1529C > T and c.2975T > G mutations, minisequencing reactions were performed with the ABI PRISM SNaPshot Multiplex Kit, in accordance with the protocol provided by the manufacturer (Applied Biosystems). The minisequencing primers were specifically designed (Table II).
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Results |
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Informativity tests
The informativity of all couples requesting PGD for NF1 was studied for the different markers; results are shown in Table I. Couple 1 was informative for the four markers and the haplotype linked to the unknown mutation could be established through material of the chorionic villus samples (CVS) of two affected fetuses. Couple 2 was informative for D17S841, IVS27AC28.4 and D17S1800; the phase was determined by studying the DNA of the patient's parents. The third couple was informative for all markers, but the segregation could not be established because the mutation appeared de novo in the male, and the couple had no previous pregnancies. Once the duplex PCR of the c.2975T > G mutation and IVS27AC28.4 was developed at the single-cell level, it was applied on single spermatozoa in order to establish the linkage between the mutation and the marker. Couple 4 was informative for IVS27AC28.4, IVS38GT53.0 and D17S1800; the phase study was done on the CVS material of an affected fetus. Couple 5 was informative for D17S841, IVS38GT53.0 and D17S1800. They had an unaffected child and DNAs of five family members were available to perform the linkage studies. The sixth couple was informative for IVS27AC28.4, IVS38GT53.0 and D17S1800, and a CVS was available to perform the linkage study.
Amplification of lymphoblasts from heterozygous individuals
The amplification, allele drop out (ADO, random non-amplification of one of the two alleles present in an heterozygous single-cell sample) and contamination rates obtained during the pre-clinical tests on single lymphoblasts are shown in Table IV. One of the protocols, the triplex PCR for D17S841, IVS38GT53.0 and D17S1800, was adapted for the analysis on the ABI PRISM 3100, but the workup on single lymphoblasts was not repeated.
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Clinical cycles and re-analysis of non-transferred embryos
The results of the clinical cycles are summarized in Table V. The six couples underwent in all 16 cycles. In four cycles, no embryos were transferred and in the remaining 11 cycles the patients had transferred one, two or three embryos, resulting in five raised levels of HCG. The pregnancy of couple 2 ensued after transfer of cryopreserved embryos. Four surplus embryos of the two PGD cycles were cryopreserved and three of them were transferred in a third cycle. The third cycle of couple 4 resulted in a biochemical pregnancy (raised level of HCG, but no fetal heart beat), and the fifth cycle in miscarriage during the eighth week. The second cycle of couple 6 resulted in a biochemical pregnancy. The pregnancy of couple 2 resulted in the birth of a healthy unaffected boy of 2370 g and 45 cm. The pregnancy of couple 3 is still ongoing and no prenatal diagnosis has been carried out.
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Non-transferred and non-cryopreserved embryos were re-analysed to verify the diagnosis during the PGD, except the embryos of the fourth cycle of couple 4, for which the PCR failed due to an operator error. All re-analysed embryos revealed that they had been correctly diagnosed during the PGD, except one embryo in the first cycle of couple 2, the embryo in the first cycle of couple 6 and one embryo in the first cycle of couple 5. In the first case, the embryo was diagnosed as affected, but the re-analysis showed recombination that had not been detected during the PGD. In the second case, the embryo had been diagnosed as abnormal due to the amplification of only the unaffected maternal allele of IVS38GT53.0 and no amplification of the mutation, but the re-analysis revealed an unaffected genotype. In the case of the first cycle of couple 5, an embryo was diagnosed as haploid during the PGD (only the maternal affected allele for the three markers was present) but the re-analysis revealed a diploid affected genotype.
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Discussion |
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NF1 is a prevalent autosomal dominant neurocutaneous disorder predisposing to cancer; 10–20% of the neurofibromas develop into malign peripheral nerve sheath tumours (Upadhyaya et al., 2004
). Over a period of 6 years 15 couples requested information concerning PGD. So far, six of them followed through, and either multiplex-PCR-based single-cell assays with linked polymorphic or mutation-specific duplex assays were developed and used.
Successful PGD depends on being able to efficiently and precisely determine the genotype of an embryo using one or two of its cells. The technical advantages of multiplex linked-marker single-cell protocols include the immediate detection of contamination, ADO and recombination leading to the decrease of the risk of misdiagnosis (Pickering et al., 1994; Findlay et al., 1995). Moreover, a diagnosis based on linked markers can theoretically be applied to more than one couple because the presence or absence of the mutation will be indirectly detected through a more generally applicable protocol.
One of the causes of misdiagnosis in PGD can be contamination. Contamination through carry-over or with extraneous genomic DNA will occur with the same probability in the negative controls as in the sample. As a consequence, after single-cell PCR, it can be unsafe to assume that the presence of a specific PCR product is the result of the amplification of the DNA of the target cell, even if the negative controls of the PCR do not contain amplified products. However, if a marker is coamplified with the mutation, or a multiplex for markers is performed, contamination with alleles not related to the samples will be evident. A further advantage of using markers is that they effectively fingerprint the contamination, allowing its source to be traced.
A second source of error linked to the single-cell PCR is ADO. This is particularly of concern in PGD for autosomal dominant disorders, where ADO of the affected allele could lead to the transfer of an affected embryo. ADO for one of the loci of a multiplex PCR will be detected through comparison with the result of the other loci.
Navidi and Arnheim (1992) and Lewis et al. (2001) demonstrated in a theoretical model that an increase in the number of studied genotypes in an embryo reduces the probability of misdiagnosis, either through the use of multiplex PCR and/or the analysis of two cells per embryo instead of one. Analysing three genotypes in a single reaction, as is performed in the multiplex PCRs reported here, may allow an accurate diagnosis on a single embryonic cell. Whether biopsy of a single blastomere causes less harm to the embryo than the biopsy of two blastomeres remains to be shown (Van de Velde et al., 2000).
Finally, when using this approach, it should be taken into consideration that recombination can occur, which could lead to misdiagnosis. To be able to detect recombination, it is important to use flanking markers. This was proven to be important since, during the PGD cycles, we were confronted with four cases of recombination that were confirmed during the post-PGD re-analysis (data not shown). It is noteworthy that recombination always occurred between one of the intragenic markers and D17S1800.
An important clinical advantage of multiplex PCRs for linked markers is the immediate availability of a PGD protocol, regardless of the specific causal mutation. Therefore, intragenic and extragenic linked markers spread over the NF1 region were first analysed for their informativity in the families requesting PGD. Three different multiplex PCRs were developed for the most informative markers and were applicable for four out of six families. For the other two (couples 3 and 6), single-cell assays for the mutation and a marker had to be developed. In couple 3, no DNA from affected family members was available to establish the phase and here too, a single-cell assay combining the mutation and a marker had to be developed. Single-sperm PCR was carried out afterwards to distinguish affected from unaffected haplotypes. In the case of couple 6, a mutation-specific protocol was developed because the affected woman was only informative for markers on one side of the mutation in NF1. The use of a marker-based protocol in this case would raise the risk of misdiagnosis because when using markers on just one side of the mutation, a recombination event between the mutation and the markers would not be detected during the PGD. To maximize the chances of finding flanking informative markers, a larger number of polymorphic microsatellite markers were searched for in the literature and databases, and new multiplex-protocols are being developed.
A previous report on PGD for NF1 describes a combination of a mutation-specific analysis with three markers: two restriction fragment length polymorphisms in exon 5 and intron 19A and a microsatellite polymorphism in intron 27A (Verlinsky et al., 2002). In this report, the use of three markers will reduce the risk for misdiagnosis through detection of contamination and ADO, but their applicability for other families will be limited. This can be argued mainly for two reasons. First, two of the used markers were restriction fragment length polymorphisms, which are not so informative as microsatellite markers. Secondly, the markers do not span the entire gene. Taking into account that an accurate test requires at least two flanking markers, couples with mutations located upstream of exon 5 or downstream of exon 29 cannot be helped with the markers used in this protocol.
The final aim of our work is to develop more single-cell assays for more markers combined in various multiplex assays in order to help most of the couples within a reasonable time span.
To date, we have performed 16 clinical cycles. In couple 1, during the second cycle there were no embryos suitable for biopsy and the third cycle was cancelled due to a bad response to the ovarian stimulation. In the 14 cycles where PGD was carried out, a diagnosis could be established in 66 of the 70 biopsied embryos (94.3%), which is a very satisfactory efficiency rate. In 11 cycles, a total of 20 embryos were transferred, resulting in a mean of 1.8 embryos per transfer. This resulted in five positive HCGs, two of these resulted in biochemical pregnancies and one in a miscarriage during the eighth week. The first pregnancy resulted in the birth of an unaffected healthy boy and the second pregnancy is still ongoing.
Though three of the re-analysed embryos had been misdiagnosed during PGD, these errors are considered acceptable since none of them could have led to the transfer of an affected embryo. In the first case, the embryo was diagnosed as affected based on weak results for IVS27AC28.4 and D17S841 and a clear result of affected for D17S1800. The re-analysis showed an unaffected pattern for IVS27AC28.4 and D17S841, and affected for D17S1800, revealing that recombination had occurred between the first two markers and the third. For the two last embryos, the misdiagnosis can be explained by the frequent occurrence of mosaicism in human preimplantation embryos. Because of our conservative diagnosis policy, we tend to diagnose embryos as affected or abnormal more easily than unaffected, especially if the genotype raises the smallest doubt. For instance, during the first cycle of couple 6, the embryo was diagnosed as abnormal since only one allele out of four had amplified. In the re-analysis it showed a normal unaffected pattern. Although this conservative diagnosis policy may reduce the number of embryos diagnosed as unaffected and suitable for transfer, this is always preferable to the risk of transferring an affected embryo. It is important to keep in mind that most of our patients choose PGD to avoid therapeutic abortion and many of them have already undergone one or more terminations.
To summarize, in the present work we discuss the development and clinical application of five PGD protocols for NF1. Two of these are mutation-specific protocols, whereas the remaining three are marker-based multiplex PCRs that can be used for different couples, which optimizes our ability to offer PGD to the patients.
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Acknowledgements |
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This work was supported by grants from the Fund for Scientific Research—Flanders and by the University Research Council. The authors wish to thank the medical, paramedical and technical staff of the Research Centre Reproduction and Genetics the referring practioners, the genetic labs that provided us with the reports on the molecular genetics of our patients and M.Whitburn from the Language Education Centre who edited the manuscript.
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Submitted on January 14, 2005; resubmitted on March 9, 2005; accepted on March 17, 2005.
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