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Preimplantation genetic diagnosis (PGD) for inherited disorders is presently applied for more than 300 different conditions. The most frequent PGD indication is cystic fibrosis (CF), the largest series of which is reviewed here, totalling 404 PGD cycles. This involved testing for 52 different CFTR mutations with almost half of the cases (195/404 cycles) performed for ΔF508 mutation, one-quarter (103/404 cycles) for six other frequent mutations and only a few for the remaining 45 CFTR mutations. There were 44 PGD cycles performed for 25 CF-affected homozygous or double-heterozygous CF patients (18 male and seven female partners), which involved testing simultaneously for three mutations, resulting in birth of 13 healthy CF-free children and no misdiagnosis. PGD was also performed for six couples at a combined risk of producing offspring with CF and another genetic disorder. Concomitant testing for CFTR and other mutations resulted in birth of six healthy children, free of both CF and another genetic disorder in all but one cycle. A total of 96 PGD cycles for CF were performed with simultaneous aneuploidy testing, including microarray-based 24-chromosome analysis, as a comprehensive PGD for two or more conditions in the same biopsy material.
Preimplantation genetic diagnosis (PGD) for inherited disorders is now applied routinely for more than 300 different conditions. One of the first and currently most frequent PGD indications is cystic fibrosis (CF), for which 404 PGD cycles were performed in this centre. This involved testing for 52 different CF mutations, which has been extremely accurate in all but one case, due to an allele-specific amplification failure that was not sufficiently appreciated at the initial stage of PGD introduction. Almost three-quarters of these PGD cycles were for seven frequent mutations, including ΔF508, and only a few for the remaining 45 CFTR mutations. The experience of 44 PGD cycles for 25 affected homozygous or double-heterozygous CF patients, 18 male and seven female partners, is described, which involving the testing simultaneously for three mutations, with no misdiagnosis, resulting in birth of 13 healthy CF-free children. Also unique data on PGD for six couples at a combined risk of producing offspring with CF and another genetic disorder is presented, which resulted in birth of six healthy children, free from both CF and another genetic disorder. Because of the advanced reproductive age of some patients, 96 PGD cycles for CF were performed with simultaneous aneuploidy testing, including microarray-based 24-chromosome analysis, in an attempt to improve reproductive outcome.
Single-gene disorders are the first group of indications for which preimplantation genetic diagnosis (PGD) was originally introduced with the purposes of performing genetic testing before pregnancy and establishing only unaffected pregnancies with no need for pregnancy termination, which is the major limitation of traditional prenatal diagnosis (
). This paper reviews this study centre’s experience of PGD for CFTR, with emphasis on CF-affected patients and couples at risk of producing offspring with CF in addition to another genetic disorder.
As will be seen, all the available approaches for PGD have been used in this study centre, including polar body (PB) analysis, blastomere and blastocyst biopsy, which were chosen depending on the indications, number of alleles tested, the parental origin of the mutation and special circumstances in each particular case, but none of these approaches can be universal. The major advantage of the PB approach is that no embryo material is removed, with less potential impact on embryo viability. Another extremely important fact concerns the accuracy, as the emphasis is on the preselection of embryos that originate from oocytes with heterozygous PB1, excluding the possibility of misdiagnosis due to undetected allele drop out (ADO), as both alleles are present. So detection of a heterozygous PB1 and a corresponding affected PB2 allows avoidance of the risk of transferring an affected embryo. Another point of value is that ADO is significantly lower in PB than in blastomeres (
) if the oocytes with the homozygous affected PB1 have to be selected. The PB approach is also of special value in couples with an affected paternal partner, as the diagnosis may be limited to a preselection of mutation-free oocytes. Finally, as will be demonstrated, concomitant testing for more than one genetic disease, especially with aneuploidy testing, is also needed. For such couples, it is useful to test for one of the conditions by the PB approach, concentrating on the others requiring testing by embryo biopsy, as simultaneous testing for many alleles in the same reaction is less accurate.
As for embryo biopsy, it is of course a more widespread approach because both paternal and maternal alleles may be tested. It is also the only possible approach for HLA typing, despite blastomere analysis being prone to error due to higher ADO and mosaicism. Although blastocyst biopsy helps to overcome the blastomere testing limitations to some extent, because a few biopsied cells are tested, it may lead to frozen–thawed embryo transfer in most cases.
As will be presented, combined PB and embryo testing is frequently required, with embryo biopsy being used also as additional confirmatory test. Also, the PB approach may not allow for selection of a sufficient number of embryos for transfer, so fertilization of affected oocytes by normal spermatozoa may still provide heterozygous unaffected embryos for transfer.
The other important methodological approach used in the study was the use of several linked polymorphic markers, introduced over 15 years ago, with the evidence for accuracy of the approach published extensively, as recommended by PGDIS and ESHRE guidelines (
Table 1 presents this study centre’s overall experience of 404 PGD cycles for CF, which is one of the world’s largest experiences for one centre. A total of 57 of these cycles were performed by PB analysis alone, resulting in the detection of 86 embryos originating from mutation-free oocytes (approximately 2.0 on average) and transferred in 44 (77.2%) cycles, which resulted in 16 (36.4%) pregnancies and birth of 16 healthy children.
Table 1Clinical outcome of 404 PGD cycles for cystic fibrosis.
In addition, 116 cycles were tested by PB analysis also involving sequential embryo biopsy for detection of unaffected carrier embryos derived from the mutant oocytes. A total of 211 unaffected embryos were identified for transfer (approximately 2.0 on average) in 99 (85.3%) cycles, which resulted in 50 (50%) pregnancies and birth of 56 healthy children.
The remaining 231 PGD cycles were performed by blastomere or blastocyst biopsy, resulting in transfer of 388 unaffected embryos in 202 (87.4%) cycles, which resulted in 106 (52.5%) pregnancies and birth of 103 (51.0%) healthy children.
Overall, of 404 cycles performed for 265 patients at risk of producing offspring with CF or CF combined with another genetic disorder, 345 cycles resulted in preselection and transfer of 685 unaffected embryos (approximately 2 per cycle), yielding 172 unaffected pregnancies (49.9% per transfer) and birth of 175 apparently healthy children.
Therefore, the clinical outcome of PGD for CF was comparable or even more favourable than routine IVF, despite transferring only one or two embryos per cycle on average. Of 175 children born following the procedure, only one misdiagnosis was observed during the whole period of over 20 years (see below). Assuming that overall experience involved the genetic testing of thousands of oocytes and embryos and resulted in preselection and transfer of 685 unaffected embryos in 345 cycles, the applied technique may be considered to be highly accurate and reliable.
In fact, a similarly high PGD accuracy was reported in this study centre’s overall series of 3056 PGD cycles for monogenic disorders, resulting in preselection and transfer of 4454 unaffected embryos in 2359 cycles and yielding 1063 (45.1%) unaffected pregnancies and birth of 1018 healthy children with only three misdiagnoses observed (including the one already mentioned) (
). Clinical outcome data is also available for 15,885 PGD cycles, collected from 39 different centres by the ESHRE PGD Consortium, which resulted in 2881 (18.1%) clinical pregnancies per initiated cycle and birth of 4227 healthy children overall, although with much lower but acceptable PGD accuracy (
This study centre performed PGD for 52 different mutations in CFTR (Table 2), usually simultaneously with strongly linked polymorphic markers, which may realistically be selected from a set of 11 markers (Figure 1). Almost half of these (195/404 cycles) were performed for ΔF508 mutation, one-quarter (103/404 cycles) for six other frequent mutations (W1282X, R117H, G551D, G542X, N1303K, 1717–1G>A), and only a few for each of the remaining 45 CFTR mutations (Table 2).
Table 2List of CFTR mutations for which PGD was performed.
As shown in Table 3, close to a half of these PGD cycles (180/404) were performed for 122 couples with the same mutation in both parents, including one with both partners carrying 1–3120G>A, two with both partners carrying W1282X, and 119 with both partners carrying the ΔF508 mutation. The remaining PGD cycles were performed for couples with two or more different mutations in partners, although the majority of cases had the ΔF508 mutation. An example of PGD for simultaneous testing for two different CFTR mutations is presented in Figure 2. As mentioned, the only misdiagnosis was observed at the initial stage of PGD, which was performed for a couple with mutation known for only one of the partners (ΔF508). Because PGD was based on testing for ΔF508 mutation only, apparently the ADO of this locus in the biopsied blastomere led to the transfer of an affected double-heterozygous embryo (
As shown in Table 3, 44 PGD cycles were performed on CF-affected patients based on the testing for three mutations simultaneously, as demonstrated in Figure 3. PGD for this case seemed to be the only choice, as the male partner was double heterozygous for ΔF508 and R117H and the female partner was a carrier of the ΔF508 mutation. To avoid testing for two different mutations simultaneously in the same blastomere, taking into consideration a risk of ADO of up to 20% for each of the alleles tested (
). As a result of testing for ΔF508 maternal mutation, performed simultaneously with multiple closely linked markers, four mutation-free oocytes were detected from the eight oocytes available for testing. Two of these embryos resulting from oocytes 3 and 4 with acceptable development potential were transferred, yielding a twin pregnancy and birth of two healthy baby girls that were confirmed to be unaffected carriers of the paternal mutation. So PGD is of special value for couples with homozygous and double-heterozygous affected partners that have only a 50% chance of having an unaffected child. The first PGD for a couple with an affected compound heterozygous male partner and female partner carrying a third mutation was performed for phenylketonuria (
With progress in the treatment of some genetic disorders, PGD will have an increasing impact on the decision of affected and well treated patients to reproduce. For example, life expectancy has been significantly improved for such common conditions as CF and thalassaemia, which represent the most frequent indications in PGD practice. In each of such cases, the strategy depends on whether the affected partner is male or female, because testing may be entirely limited to oocyte testing if a male partner is affected, in contrast to embryo testing, which will be required if the female partner is affected, as illustrated in Figure 4, involving the testing for the two different CFTR mutations. Because the female partner was double heterozygous, involving two different mutations (R117H and G542X), and the male partner was a carrier of R117H mutation, PGD was based on blastomere biopsy. To be able to test simultaneously for the required number of the linked markers, the paternal and maternal haplotypes were first established, paternal using single-sperm PCR and maternal by PB1 analysis. As can be seen from the pedigree, the couple had two previous pregnancies, the first resulting in spontaneous abortion of twins, with the second terminated following prenatal diagnosis of affected fetus with CFTR mutation. Multiplex PCR for the two mutations in CFTR in this case was combined with age-related aneuploidy testing because of the mother‘s advanced reproductive age. Multiplex hemi-nested PCR analysis was performed on single blastomeres from 14 embryos, allowing simultaneous detection of the paternal and maternal CFTR haplotypes and non-syntenic short tandem repeats located on chromosomes 13, 16, 18, 21, 22 and XY. Six embryos were predicted to be unaffected CFTR mutation carriers, based on the presence of the normal paternal CFTR. In addition to avoiding the transfer of affected double-heterozygous embryos, three aneuploid embryos were identified, including aneuploidy for chromosomes 13, 18, 21 and X, which were also excluded from transfer and freezing. Two unaffected embryos (nos. 6 and 7) were transferred, resulting in an unaffected pregnancy and birth of a healthy baby girl, confirmed to be a carrier of the maternal G542X mutation. DNA analysis of the newborn baby revealed a genetic profile identical to that of embryo 7, showing the usefulness and accuracy of combined mutation, linkage and aneuploidy testing in PGD for single-gene disorders in patients of advanced reproductive age.
Overall, of 44 PGD cycles performed for 25 CF-affected patients, including seven female and 18 male partners, 72 unaffected embryos were identified for transfer in 36 cycles, resulting in 17 clinical pregnancies and births of 13 unaffected children with no misdiagnosis (two pregnancies still ongoing and 2 pregnancy losses) (Table 3). So PGD for CF-affected patients in this experience is as efficient and highly accurate as for CF carrier couples.
Concomitant PGD for CF and other Mendelian conditions
A total of 11 PGD cycles was performed for CF in six couples also at risk of producing the offspring with other genetic conditions, including Darier disease, facioscapulohumeral muscular dystrophy (FSHD), Aicardi-Goutieres syndrome, hereditary haemochromatosis and Robertsonian translocations. Unaffected embryos were available for transfer in all the cycles and the transfer of a total of 14 embryos resulted in seven pregnancies and birth of six children free of both CF and the other genetic condition.
One of these cases is presented in Figure 5, which presents the results of PGD for the ΔF508 mutation combined with testing for the Darier disease de-novo mutation in ATP2A2. PGD was based on sequential PB1 and PB2 testing for both mutations, followed by blastomere biopsy of the embryos containing the CFTR mutation for possible detection of unaffected carrier embryos. Of 15 oocytes tested by sequential PB1 and PB2, only two oocytes (nos. 9 and 14) appeared to be free of both mutations, so the embryos resulting from these oocytes were transferred, resulting in a singleton pregnancy and birth of a healthy baby girl, free of both diseases. A sequential blastomere analysis of the four embryos resulting from oocytes with the ΔF508 mutation and without the deletion in ATP2A2 (one of the tested embryos was without PB results) allowed preselection of one embryo (no. 2), a carrier of the ΔF508 mutation, which was frozen for future use by the couple.
A sequential PB1 and PB2 analysis, followed by blastomere biopsy, was also applied for combined testing for ΔF508 and FSHD, and ΔF508 and hereditary haemochromatosis (C282Y mutation in HFE). PGD in the former case resulted in detection of two of eight embryos free of FSHD deletion for transfer, which yielded a singleton pregnancy and birth of an unaffected carrier of the ΔF508 mutation free of FSHD. PGD in the latter case allowed preselection of two of five embryos, one without the C282Y mutation but a carrier of ΔF508, and one resulting from a mutation-free oocyte (data not shown). Two PGD cycles were performed for CFTR mutation and Aicardi–Goutieres syndrome by trophectoderm biopsy, which resulted in an ongoing unaffected pregnancy free of ΔF508 and Aicardi–Goutieres syndrome. A PGD cycle for two CFTR mutations combined with testing for spinal muscular atrophy (SMA) is presented in Figure 6, which also involved an additional aneuploidy testing (to be discussed further).
A concomitant PGD for two different genetic disorders has previously been described in a childless Ashkenazi Jew couple at risk of producing offspring with Tay Sachs and Gaucher disease, with both parents carrying two different mutations in β-hexosaminidase A and β-glucocerebrosidase genes (
). Six embryos were analysed, of which one was wild type for both Tay Sachs and Gaucher disease, three were wild type for Gaucher disease and carrier of Tay Sachs, and two were compound heterozygote for Tay Sachs. Two of the four transferable embryos which developed into blastocysts were transferred, resulting in a singleton pregnancy and birth of a healthy child free of both conditions.
The other concomitant PGD was performed for Charcot-Marie-Tooth and Fabry diseases in a couple with both partners carrying the Fabry mutation and the male partner with Charcot-Marie-Tooth disease (
). As in PGD for CF-affected male partners, testing was performed by a sequential PB1 and PB2 analysis of Fabry disease, followed by embryo biopsy and testing for Charcot-Marie-Tooth, which allowed the identification and transfer of an unaffected embryo and resulted in a triplet pregnancy and birth of three healthy children, which appeared to be monozygotic triplets. Although the mechanism for the formation of monozygotic triplets in this case is not understood, the data showed that concomitant PGD for more than one condition is feasible and may be performed using the combination of different biopsy techniques, allowing the accurate detection of both conditions.
A similar approach was used for other concomitant PGD cycles for BRCA1 and SMA, and BRCA2 and MEN1 (
). Testing for both mutations in each of these cycles allowed identification of two unaffected embryos for transfer in both cases.
PGD for CF with concomitant 24-chromosome aneuploidy testing
This study centre’s experience of concomitant PGD for CFTR together with aneuploidy testing includes 96 cycles, resulting in preselection of 153 euploid unaffected embryos for transfer in 92 cycles and yielding 34 (37.0% per transfer) clinical pregnancies and birth of 32 unaffected children (Table 4). Aneuploidy testing was initially performed by fluorescence in-situ hybridization or PCR (
), but the procedure became more robust with the introduction of microarray-based technology, which makes possible comprehensive testing for multiple gene mutations, linked markers and aneuploidy in the same biopsy material. This is becoming particularly important with the presently increasing number of PGD patients at risk of age-related aneuploidies.
Table 4PGD for cystic fibrosis combined with aneuploidy testing.
The centre used an array comparative genomic hybridization (CGH) platform, developed by BlueGnome Cambridge, UK because it can be applied to all biopsy materials, including PB1, PB2, blastomeres and blastocyst, it allows completion of the test within 12 h and it provides an accurate result in over 90% samples. In brief, this protocol consists of at least five steps, including amplification (2 h), labelling (2.5 h), hybridization (3.5 h), washing (30 min), scanning (30 min) and data analysis (1 h).
One of the critical steps of the procedure is whole-genome amplification with the Super Plex Single Cell Whole Genome Amplification Kit, which is performed according to the manufacturer‘s instructions. Specific quality-control criteria for sample quality and quantity are used to ensure that only specific amplifications are labelled. The fluorescent labelling system is used for the labelling of the amplified samples from biopsy materials as well as for the labelling of a commercially available reference DNA. Test and reference DNA coprecipitation, their denaturation, array hybridization and the post-hybridization washes are performed according to the protocol provided by the manufacturer.
Therefore, whole-genome amplification is the first step in the procedure for 24-chromosome testing, followed by testing for CFTR mutations in the amplification products. The technique tests all 24 chromosomes for any gain or loss with the bacterial artificial chromosome pooling strategy, which coupled with the uniquely designed software enables straightforward results on aneuploidy to be obtained in a single cell. Currently, two BlueGnome 24sure array formats are used for two applications. Bacterial artificial chromosomes spotted on the 24sure array are selected on the basis of having little variation in over 5000 hybridizations, and so deliver the highest level of reproducibility and sensitivity in aneuploidy testing. A laser scanner is used to excite the hybridized fluorophores and to read and store the resulting hybridization images, using the special software provided by BlueGnome.
The first case of such an approach was reported by
and there is presently an increasing number of PGD using microarray-based 24-chromosome aneuploidy testing in many centres as a basis for combined PGD for single-gene disorders and 24-chromosome testing (
). This study centre’s present experience of 24-chromosome aneuploidy testing together with PGD for single-gene disorders and HLA typing includes 106 cycles for the following inherited disorders: achondroplasia, Aicardi–Goutieres syndrome (already discussed), arthrogryposis, BRCA1, Duchenne muscular dystrophy, dystonia, DYT1, familial hyperinsulinism, fragile-X syndrome, Gaucher disease, GM1 gandliosidosis, Holt–Oram syndrome, Huntington disease, Marfan syndrome, mosaic variegated aneuploidy, myotonic dystrophy, neurofibromatosis type 1, Niemann–Pick disease, OCA1 and OCA2, polycystic kidney disease, SMA, tuberous sclerosis (data not shown) and CF.
A concomitant PGD for SMA, ΔF508 and array CGH-based 24-chromosome aneuploidy testing is presented in Figure 6. As seen from this cycle, both partners are carriers of SMN1, while father is also a carrier of ΔF508 and mother also the carrier of the N703S CFTR mutation. So sequential PB1 and PB2 testing was performed first to detect oocytes free of CFTR and SMN1 mutations, with further testing of the resulting embryos by 24-chromosome aneuploidy. Of the five oocytes available for testing, only one oocyte (no. 1) appeared to be free of both SMN1 and N703S mutations, while others were with either a CFTR or SMN1 mutation. Testing of the resulting embryos confirmed unaffected status of the embryo resulting from oocyte 1, which was also 24-chromosome aneuploidy free. The embryos resulting from oocytes 3 and 7, although unaffected carriers of CFTR and SMN1 mutations, appeared to be with monosomy 5 (embryo 3) and trisomy 10 and monosomy 16 (embryo 7). The embryo resulting from oocyte 5 was affected for SMA and a carrier of ΔF508 in CFTR. Finally, the embryo resulting from oocyte 6 appeared to be an unaffected carrier of ΔF508 and free of aneuploidy. Therefore, two embryos (embryos 1 and 6) were transferred, resulting in a twin pregnancy and birth of two unaffected carriers of SMA and CF.
Overall, a combined comprehensive approach for 24-chromosome aneuploidy testing, together with PGD for single-gene disorders and preimplantation HLA typing, resulted in transfer of 153 unaffected aneuploidy-free embryos in 82 of 96 initiated cycles, resulting in 34 pregnancies (41.4% per embryo transfer) and birth of 32 healthy children, suggesting the usefulness of this approach in an effort to optimize the pregnancy success in patients of advanced reproductive age referring for PGD.
The presented data demonstrate an extremely high accuracy of PGD for CF, irrespective of testing for one, two or three CFTR mutations simultaneously, and also concomitant testing for another genetic disorder. It is also practical to combine PGD for CF and another genetic disorder with microarray-based 24-chromosome aneuploidy testing as a possible comprehensive PGD for multiple indications in patients of advanced reproductive age.
Eldar Geva T.C.
Simultaneous preimplantation genetic diagnosis for Tay-Sachs and Gaucher disease.
Declaration: The authors report no financial or commercial conflicts of interest.
Dr Svetlana Rechitsky is a graduate of Kharkov University’s genetics faculty and received her PhD in experimental molecular embryology from the 2nd Moscow Medical Institute in 1986. She moved to the Reproductive Genetics Institute in 1989, where she heads the DNA laboratory, which has performed the largest PGD series for single-gene disorders, including designing the analysis for the majority of them. She has published more than 60 papers in the field of PGD, including a key contribution to the Atlas of Preimplantation Genetic Diagnosis, and chapters on single-gene disorders for both editions of Practical Preimplantation Genetic Diagnosis.