Introduction: Prcimplantalion genetic diagnosis (PGD) was introduced in the nineties of last century to prevent terminations of pregnancy for fetuses affected by monogenic diseases. Embryos obtained in vitro are biopsied, and the biopsied blastomeres are used to establish a genetic diagnosis, after which only the embryos free of the disease under consideration are transferred into the womans womb. In this presentation, a brief history of technical evolutions in PGU for monogenic diseases will be outlined, followed by an overview of our own and international results.
Materials and methods: All tests used to dale are PCR-derivcd: the earlier PGDs involved two rounds of PCR followed by analysis on ethidiumbromide-stained gels. this type of tests shows a high rate of inaccuracy mainly due to ADO, which led to two documented misdiagnoses. Because of this, it was replaced at the end of the nineties by fluorescent PCR followed by analysis on automated sequencers. In fluorescent PCR, the primers used to initiate the PCR reaction are labelled with a fluorochrome, so that the PCR products obtained are also fluorescently labelled, "these PCR products can then he used for fragment analysis on an automated DNA sequencer. Fragment analysis can be carried out directly for e.g. short deletions or insertions, or for point mutations after cutting with restriction en/ymcs. Because of the high efficiency of fluorescent PCR as compared to non-fluorescent PCR, the ADO rate is significantly lower, and thus the misdiagnosis risks were substantially decreased. Fragment analysis is also useful for PCiD using linked microsatellite markers. The combination of mutation analysis combined with linked markers was the next important development that led to a more accurate diagnosis, because now ADO could be determined. More recently, other types of DNA analysis already in use in routine DNA diagnosis, such as minisequencing and sequencing were introduced in PCiD.
Finally, (lie latest development in single cell analysis is (lie use of whole genome amplification. Because the total DNA from one blastomere can be amplified to DNA at the micrograin level, this pre-amplified DNA can then be used for more than one application, e.g. for karyotyping with comparative genomic hybridisation (CCil-1) and a monogenic disease with mutation and several markers simultaneously.
Results and discussion: At our centre, we have a large experience with different types of PCiD for monogenic diseases, and illustrative examples will be given during the presentation, (lie overall experience in PGD for monogenic diseases of the Centres for Medical Genetics and Reproductive Medicine of the A7-VLJB will be shown. International data as obtained through the LSHRR PGD Consortium will also be discussed. In conclusion, PGD is constantly evolving as far as (lie genetic analysis part is concerned, and closely follows the new technologies used in oilier brandies of DNA analysis. It is to be hoped that further technical improvements will allow practitioners to obtain all necessary results from just one biopsied blastomere. thus insuring that the viability of the embryo is secured as much as possible.