Preimplantation genetic diagnosis for beta-thalassaemia using sequencing of single cell PCR products to detect mutations and polymorphic loci

Abstract

In order to carry out preimplantation genetic diagnosis (PGD) for beta-thalassaemia, we have applied direct sequencing of single cell PCR products to detect mutations and polymorphic loci within the beta-globin gene. Conventional duplex PCR was used to amplify two regions of the beta-globin gene with an amplification efficiency of 79% for blastomeres. Sequencing data were obtained for 100% of amplified products, with 12% having confirmed allele drop-out (ADO). A double ADO event was observed at least twice, confirming the real risk of such an event during PGD. In one couple, the presence of a polymorphism linked to the female partner's mutation enabled us to eliminate the risk of misdiagnosis due to double ADO without having to amplify both mutations within the same PCR product. We present here the data from eight clinical PGD cycles for three couples resulting in a singleton pregnancy and a twin pregnancy with all babies confirmed to be free from beta-thalassaemia (major).

Real-time PCR for single-cell genotyping in sickle cell and thalassemia syndromes as a rapid, accurate, reliable, and widely applicable protocol for preimplantation genetic diagnosis

“…The trials using single lymphocytes from an individual heterozygous for a b-thalassemia mutation and compound heterozygous at both microsatellite loci, demonstrated that the optimized protocol had a PCR success of almost 99% and an ADO rate of around 1% for all loci. These results compare favorably to all previous methods described for PGD for hemoglobin disorders, which report PCR success ranging between 87 to 99% and ADO of 0 to 12% for the b-globin gene locus [Holding and Monk, 1989;Monk and Holding, 1990;Varawalla et al, 1991;Ray et al, 1996;El-Hashemite et al, 1997;Kuliev et al, 1998;Vrettou et al, 1999;De Rycke et al, 2001;Piyamongkol et al, 2001;Hussey et al, 2002], and they are well within the limits generally set by centers offering PGD [ESHRE PGD Consortium Steering Committee, 2002]. As mentioned above, the occurrence of ADO will not lead to transfer of an affected embryo by the real-time genotyping method, although it may lead to risk of rejecting a heterozygous embryo.…”

“…In the cohort of blastomeres that were analyzed, a b-globin genotype was achieved in 92% of cells, again comparing favorably to previous reports of clinical PGD cycles [Ray et al, 1996;Kuliev et al, 1998;Kanavakis et al, 1999;De Rycke et al, 2001;Hussey et al, 2002] and improved relative to a rate of 78% PCR success when genotyping single blastomeres using our previously described nested PCR/DGGE method TraegerSynodinos et al, 2003]. It is more difficult to evaluate ADO in clinical PGD cycles, unless it presents as an impossible b-globin genotype based on the parental mutations in each cycle.…”

“…The method also proved to be very flexible relative to the variation of b-globin genotype interactions analyzed. It is potentially applicable for a very wide spectrum of interactions without any modification (Tables 2 and 3), an advantage not addressed by most of the methods that have so far been described for PGD applied to the inherited disorders of hemoglobin synthesis [Holding and Monk, 1989;Monk and Holding, 1990;Varawalla et al, 1991;Ray et al, 1996;Kuliev et al, 1998;De Rycke et al, 2001;Piyamongkol et al, 2001;Hussey et al, 2002]. The development of the described PGD protocol was in part facilitated by more than a decade of accumulated experience in single-cell PCR-based diagnostics [ESHRE PGD Consortium Steering Committee, 2002;Harper and Bui, 2002;Thornhill and Snow, 2002;Traeger-Synodinos et al, 2003].…”

“…Furthermore, extraneous contamination must be stringently avoided and ideally monitored in each individual sample to preclude unacceptable misdiagnosis [Lewis et al, 2001;Harper and Bui, 2002;Thornhill and Snow, 2002;Traeger-Synodinos et al, 2003]. Overall, an ideal PGD method should be sensitive, accurate, reliable, rapid, simple, and widely applicable, but so far none of the previously described approaches to PGD of bthalassemia have addressed all of these criteria [Holding and Monk, 1989;Monk and Holding, 1990;Varawalla et al, 1991;Ray et al, 1996;El-Hashemite et al, 1997;Kuliev et al, 1998;Vrettou et al, 1999;De Rycke et al, 2001;Piyamongkol et al, 2001;Hussey et al, 2002].…”

Well-devised quantification analysis for duplication mutation of Duchenne muscular dystrophy aimed at preimplantation genetic diagnosis

“…The remaining cases are due to point mutation (25-30%) [16] or duplications (5-10%) [5,17], but there have been no reports of PGD for DMD to the best of our knowledge. PGD for point mutation was performed for familial amyloidotic polyneuropathy (FAP) by nested PCR and single-stranded conformational polymorphism (SSCP) analysis [18], and for β-thalassaemia using sequencing of single cell PCR products [19]. Meanwhile, only a few reports exist concerning PGD for duplications [20,21], of which the detection is commonly possible using DNA probes located in the duplicated region (FISH or Southern blotting method).…”