Centres for 1 Reproductive Medicine and 3 Medical Genetics, and 4 Department of Medical Oncology and Hematology, Medical School and University Hospital, Vrije Universiteit Brussel, Belgium and 2 Genetics and IVF Unit, Department of Obstetrics and Gynecology, Medical School, University of Ioannina, Greece
5 To whom correspondence should be addressed at Centre for Reproductive Medicine, AZ-VUB, Laarbeeklaan 101, 1090 Brussels, Belgium. e-mail: hilde.vandevelde{at}az.vub.ac.be
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Abstract |
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Key words:
-thalassaemia/HLA-identical/microsatellite markers/preimplantation genetic diagnosis
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Introduction |
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Allogeneic haematopoietic stem cell transplantation (alloSCT) is the only curative treatment for thalassaemia major (Lucarelli et al., 1998, 1999, 2002). When an HLA-identical sibling marrow donor is available and in patients with effective chelating therapy prior to transplantation as well as absence of hepatomegaly or portal fibrosis (low Pesaro risk score), the chance of cure is currently
90%. In addition, alloSCT from alternative donors or donor sources has been applied succesfully in patients with haemoglobinopathies including
-thalassaemia (Gaziev et al., 2000
; La Nasa et al., 2002
; Locatelli et al., 2003
), but is less favourable compared with that with an HLA-identical donor. On the other hand, the outcome after alloSCT with HLA-identical stem cells obtained from umbilical cord seems to be comparable with bone marrow transplantation.
Preimplantation genetic diagnosis (PGD) is an established method for the diagnosis of genetic diseases at the embryonic stage so that implantation of affected embryos can be avoided (ESHRE, 2002). Couples opting for PGD to avoid the transmission of a genetic disease have to undergo an IVF treatment with ovarian stimulation and ICSI. The embryos have to be biopsied at day 3. A genetic analysis of the blastomeres is performed in order to be able to select unaffected embryos for transfer to the uterus. For monogenic diseases, PCR is applied at the single-cell level. We developed a novel PGD strategy in which the detection of a particular gene mutation was combined with the selection for HLA-identical embryos in order to obtain an unaffected child that can become an HLA-matched donor for its sibling. This strategy was validated in three different families with
-thalassaemia and may offer a new therapeutic perspective for thalassaemic patients as well as for those with other genetic diseases that can be cured with alloSCT.
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Patients and methods |
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The parents were seen by an experienced psychologist who evaluated their motivation for having another child to become a donor for their affected sibling, i.e. were the parents planning to have another child in any case, or was the plan to have another child solely for the purpose of treating the affected child? Both situations occurred and were accepted.
Case descriptions
In each -thalassaemia family, the healthy siblings were HLA-non-identical to their affected sibling. Two couples were of Italian origin and one of Turkish origin. The distinct mutations in the
-globin gene are represented in Figure 1. In the first couple, both parents are carriers of a C>T mutation in codon 39 of the
-globin gene. They have one healthy and one affected child; the mother (age 35 years) already had two miscarriages. In the second family, the disease is caused by two mutations (IVS1+6T>C and IVS1+110G>A). The mother (age 36 years) had one miscarriage, and two affected pregnancies were terminated after prenatal diagnosis (PND). The couple have an affected child and a healthy child after PGD at our centres for which HLA typing had not been done at that time (De Rycke et al., 2001
). The third couple (mother age 31 years) have one affected child and two healthy children, while two affected pregnancies were terminated after PND. The disease in this family is due to two different mutations in codon 8/9+G and IVS1+110G>A of the
-globin gene.
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Mutation analysis in single cells
Mutations in the -globin gene (Hb) were detected using primers as previously reported (De Rycke et al., 2001
): HB1/HB2, HB1/HB3 and hemi-nested PCO5/HB3/HB9 (HB9 forward: CTGACTCCTGAG GACAAG) for the three families, respectively (Figure 1). Restriction enzyme digestion was performed on 5 µl PCR product as described by the manufacturer (New England Biolabs, Westburg, Leusden, The Netherlands) (Figure 1). The C>T mutation in codon 39 in the
-globin gene abolishes an NlaIV restriction site, and the codon 8/9+G mutation creates a PflFI restriction site. The
-thalassaemia family with the IVS1+6T>C (SfaNI digests the affected allele) and IVS1+110G>A (AvaII digests the normal allele) mutations has been reported previously (De Rycke et al., 2001
).
Collection of single lymphoblasts
Lymphocytes isolated from blood samples from the couples and their offspring were transformed by EpsteinBarr virus (EBV) and cultured according to standard procedures (Ventura et al., 1988). The procedure for the collection of single lymphoblasts into 0.2 ml PCR tubes containing 3 µl of alkaline lysis buffer (ALB) [200 mM NaOH, 50 mM dithiothreitol (DTT)] (Li et al., 1988
) and blanks has been described previously (Sermon et al., 1998a
).
Multiplex PCR protocols
Prior to multiplex PCR, cells were lysed by incubation for 10 min at 65°C. The Expand High Fidelity (EHF) PCR system was used as described by the manufacturer (Roche Diagnostics, Brussels, Belgium); a hot start was included in all procedures. Optimal PCR conditions for each primer combination were set up on single heterozygous lymphoblasts. In general, two consecutive PCR rounds were performed (Table I): (i) 5 min 95°C, 10x (30 s 95°C, 30 s 55°C, 30 s 72°C), 7 min 72°C; and (ii) 5 min 95°C, 40x (30 s 95°C, 30 s 5355°C as required, 30 s 72°C), 7 min 72°C. In the first round, the PCR mix contained all primers, EHF buffer 2, 2 mM dNTP, 1.4 U of EHF DNA polymerase and neutralization buffer (10 mM Tricine, pH 8.3). For the second round, primer combinations were split up. A 3 µl aliquot from the first round was transferred into a new PCR tube, and the neutralization buffer was omitted from the reaction mix.
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Embryo biopsy
Laser biopsy was performed on the morning of day 3 (after fertilization) according to previously described procedures (Joris et al., 2003). Briefly, embryos were incubated for 5 min in Ca2+Mg2+-free medium (EB10, Vitrolife, Sweden) for decompaction. Embryo biopsy was performed in HEPES-buffered Earles medium supplemented with 0.5% (w/v) human serum albumin. Zona drilling was performed by fixing the embryo on a holding pipette and subsequently applying two or three pulses of 58 ms (1.48 µm) on the zona pellucida. The opening was made between two blastomers. Two cells were gently aspirated using a biopsy pipette (inner diameter 3540 µm) and released into the medium. Two blastomeres were taken from each embryo with at least six cells and <50% fragmentation, except in two cycles where one cell was taken because of high workload (restrictions in time and equipment). Procedures for washing, transfer into PCR tubes containing 3 µl of ALB, and blanks have been described previously (Sermon et al., 1998b
).
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Results |
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In summary, of the total number of 58 embryos tested, results of combined PCR were as follows: 54 out of 58 (93.1%) successful, one amplification failure and three inconclusive to detect the thalassaemia-related mutations. Three embryos were transferred and 50 embryos were retested. In all these embryos, the HLA typing was confirmed (100%). In six clinical cycles, two transfers were performed and one clinical pregnancy (defined as fetal heart beats on ultrasound) ensued that ended in an early miscarriage at 8 weeks.
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Discussion |
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For the majority of patients with severe -thalassaemia, blood transfusions and iron chelation therapy are the mainstays of medical treatment. Even in Western countries and despite the availability of good medical treatment,
50% of affected patients die before the age of 35 years (Modell et al., 2000
). On the other hand, the cure rate of thalassaemia with allogeneic bone marrow transplantation using HLA-identical donors is
90% in children at an early stage of the disease. The problems with alloSCT are the availability of an HLA-identical sibling donor and the transplant-related toxicity, mainly due to graft-versus-host disease. With the current status of international registries such as the National Marrow Donor Program (NMDP), it was recently shown that at least one 6/6 matched donor or umbilical cord blood unit was available for 80% of patients (Krishnamurti et al., 2003
). However, results of alloSCT using unrelated donors are less favourable compared with the use of HLA-identical donors, although experience is limited. Out of a recent series of 32 patients, and using extended haplotypes in the majority of cases, the survival was 69% with a median follow-up of 30 months and the incidence of chronic graft-versus-host disease was 25% (La Nasa et al., 2002
). Related umbilical cord blood is an excellent alternative source of stem cells for patients with haemoglobinopathies (Locatelli et al., 2003
). In a recent analysis of 44 patients reported to Eurocord, the 2-year probability of survival for patients with thalassaemia was 79% and even 89% in those belonging to a low risk category. The study also showed a very low risk of graft-versus-host disease and suggested that optimization of the transplant programmes was likely to improve the results further.
From the technical standpoint, we were faced with the difficulty of combining PGD for -thalassaemia with HLA typing on a single-cell level. PGD for HLA typing is very complex because the locus is highly polymorphic. Taking into account the size of the region (4 x 103 kb), the different regions (class I and class II), the large number of loci (A, B, C, DR, DP and DQ) and the possible recombination within the region (Thomsen et al., 1985
; Sullivan et al., 1997
), the development of a reliable single-cell PCR for each couple requesting a matched donor for their affected child would be time consuming. A direct HLA typing approach using allele-specific primers has been reported for Fanconis anaemia (Verlinsky et al., 2001
). Such a strategy that is based on the absence or presence of an allele may be susceptible to technical errors including ADO and contamination, resulting in misdiagnosis.
The use of microsatellite markers for PGD has already been shown to be an elegant methodology to improve PGD protocols for cystic fibrosis, an autosomal recessive disease for which >1000 distinct mutations are known and thus making the development of mutation-specific protocols for PGD unfeasible (Dreesen et al., 2000; Goossens et al., 2000
; Moutou et al., 2002
). Similarily, our approach for indirect HLA typing is to ascertain the inheritance of the matching haplotypes by fingerprinting the HLA locus using informative STR markers. By selecting two outer markers (a telomeric and a centromeric marker that are preferentially closely associated with the loci because recombination could be observed) and two inner markers (one within class I-A and -B and one within class II-DR and DQ) for single-cell multiplex PCR, we indirectly type the HLA locus by segregation analysis and control for recombination within the region. So far, we have not observed recombination in lymphocytes or embryos, indicating that selection of microsatellite markers was highly adequate. Informativity testing has now been done in 12 families using the bank of seven STRs; only in one family was recombination observed using the external centromeric markers. One particular combination of markers can be used in five families (our unpublished data). The reliability of this strategy is further ensured by carefully analysing in advance the limitations of each individual protocol on a large number of heterozygous lymphoblasts. The accuracy for the HLA typing was 100%; combined PGD was successful in 54 of 58 (93%) cases and the few technical failures were due to (i) unexpected low amplification resulting in no diagnosis; or (ii) the fact that ADO occurred when only one cell from the embryo could be analysed, resulting in misdiagnosis. In the latter case, misdiagnosis could have been avoided using two-cell biopsy. The debate of one- versus two-cell biopsy is still ongoing. Retrospectively we found that the implantation capacity of an embryo is not impaired when removing two cells (18% implantation rate in Van de Velde et al., 2000
). However, Pickering et al. (2003
) reported an implantation rate of 27% that may reflect their policy to biopsy only one cell. A prospective randomized controlled study has been set up at our centres to evaluate the implantation capacity after one- versus two-cell biopsy in relation to the efficiency and reliability of the test (occurrence of no diagnosis or misdiagnosis by ADO, contamination or amplification failure).
Another important advantage of our strategy is its applicability to other diseases where a similar combination of excluding a genetic disorder and selection for an HLA-identical embryo may be requested, such as sickle cell anaemia, Fanconis anaemia, Wiskott Aldrichs syndrome and severe chronic granulomatosis. By extending our experience to these indications, we were able to set up a bank of seven markers to ensure informativity in all families accepted in our programme (the feasibility of the approach has been validated now in nine different families (our unpublished data). The establishment of such an STR bank enables us to substantially shorten the waiting list to start a treatment cycle to a median of 48 weeks because a number of STR combinations have already been worked out and can be used for other families in which informativity can be ensured by using the same STRs. It is important to keep the time to develop a family-tailored PCR protocol as short as possible. Obviously there is a positive impact on overall cost-effectiveness, but there are also clinical arguments. Patients with congenital anaemias can survive for many years. However, they will develop a number of time-dependent complications such as liver fibrosis (thalassaemia) and vaso-occlusive events (sickle cell anaemia). Such complications cause direct morbidity and mortality but may also reduce the success rate of an eventual transplant procedure. For patients with congenital immune deficiencies, the continuous risk of potentially dangerous infections will decrease if the transplant is performed earlier.
We also have to consider some limitations of our technique. First, the method is labour-intensive and thus the financial cost is high. On the level of a health care programme, this aspect must be compared with the probably higher costs related to the use of unrelated donors or a continued programme of standard medical treatment (if that is an option) for a number of decades with a no-cure perspective. Secondly, the lower the number of embryos, the lower the probability of having a suitable one. The number of embryos available during a cycle is dependent on the efficacy of the ovarian stimulation protocol on the one hand and the age of the mother on the other. Theoretically, one in four embryos are HLA identical and three out of four will not carry the mutation or be heterozygous so that only three out of 16 will be transferable. Thirdly, the intrinsic implantation capacity of the embryos is highly variable and the chance of a successful pregnancy may be not higher than 26% (Van de Velde et al., 2000), leaving room for further improvement. Finally, our strategy involving embryo selection is likely to raise some ethical controversy. We argue that the selection of a healthy child by means of PGD is an accepted alternative to other techniques of PND (ESHRE, 2002
) for patients who wish to avoid offspring with a genetic disease. On the other hand, a selection based on HLA type may be more difficult to accept. One might reason that such a genetic background does not a priori infer any advantage or disadvantage for the newborn and that anyhow this genotype is inherited in 25% of cases, only by chance. In addition, the issue of instrumentalization of the child to be born may be raised: the child is created to cure another one. This difficult ethical issue has been discussed previously (Pennings et al., 2002
) and can at least be partially addressed by careful genetic and psychological counselling of the parents so that their motivation and capacity to raise another child can be fully assessed and integrated in the selection of candidates for this strategy.
In summary, we have developed a method to combine PGD for -thalassaemia and a number of monogenic diseases with the selection of HLA-identical embryos so that the unaffected child to be born can be a donor of umbilical cord blood stem cells to transplant and eventually cure an affected sibling. This novel approach that is characterized by its reliability and a relatively short work up time of development and application may represent a new and curative therapeutic option for many potential candidates, although further validation on a larger number of cases remains necessary.
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Acknowledgements |
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Submitted on October 9, 2003; accepted on December 9, 2003.