Apessos, Angela; (2000) Molecular methods in preimplantation genetic diagnosis with emphasis on the Fragile X syndrome. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Preimplantation genetic diagnosis (PGD) is a form of prenatal diagnosis. It is performed on the third day after fertilisation, on embryos of couples at risk of transmitting an inherited disorder to their offspring. In this way, only embryos without the mutation under investigation are transferred in order to initiate a pregnancy. Embryos used for PGD are generated by routine IVF procedures. Fragile X syndrome is the most common form of inherited mental retardation. The causative mutation has been identified as an expansion of a triplet (CGG)n repeat in the 5' untranslated region of the FMR1 gene. The expansion is refractory to PCR due to preferential amplification of the smaller allele in heterozygous cells and the high GC content of the repeat and surrounding sequences. Currently, the only method of diagnosis available for this disease is PCR followed by Southern blotting in order to detect the expanded allele. This, however, is not suitable for preimplantation diagnosis as it is time consuming and requires a lot of DNA. Furthermore, the time at which the amplification occurs in the embryo is not yet determined so that a test relying on detecting the expansion may not be suitable for preimplantation diagnosis. For this reason polymorphic markers linked to the mutation are used to diagnose this disorder in preimplantation embryos. Meanwhile methods for the direct detection of the mutation in single cells have been tested in order to study the timing of the expansion in embryos diagnosed as affected after preimplantation genetic diagnosis. These were applied to spare oocytes and embryos from an IVF cycle. Finally, methods for PGD of sickle cell anaemia have been tested in order to determine the most efficient one. These include restriction enzyme digestion of PCR products, single strand conformation polymorphism (SSCP) followed by staining of the DNA with the silver staining technique, and fluorescent PCR followed by SSCP on an automated fluorescent sequencer (ALF, Pharmacia).

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