Kakourou, G.; (2009) Protocol development for analysis of the DMPK repeat in preimplantation genetic diagnosis and the investigation of gene expression in human oocytes and blastocysts. Doctoral thesis , UCL (University College London).

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Abstract

Myotonic dystrophy type 1 (DM1) is an autosomal dominant disorder caused by expansion of an unstable CTG repeat within the 3’UTR of the DMPK gene, which expands further in length during transmission from generation to generation. Prenatal diagnosis is available, although the decision for pregnancy termination can be difficult due to the variable phenotypic expression of DM1. In vitro fertilisation with preimplantation genetic diagnosis (PGD), offer another reproductive option for affected couples, which involves genetic analysis and selection of an unaffected embryo to establish a pregnancy. These technologies have also provided access to human gametes and preimplantation embryos and encouraged research aimed at understanding the molecular pathways controlling human preimplantation development. The first part of this study focused on the improvement of existing techniques for PGD and the development of universal multiplex fluorescent PCR PGD protocols for the efficient and accurate diagnosis of DM1. The second part of the study involved followup analysis of DM1 affected and unaffected embryos donated for research with the aim to investigate transmission of the CTG repeat from the affected and unaffected parent to the preimplantation embryo. The final objective was to obtain a global gene expression profile by microarray analysis of human oocytes and blastocysts, with a focus on important functional pathways. The protocols developed achieved high efficiency and accuracy of diagnosis, reduced the genetic work-up time, overall supporting PGD for DM1 as an effective and practical alternative to prenatal diagnosis. This study also adds to current evidence regarding CTG repeat transmission and provides information on repeat expansion and embryo quality in DM1. A comparison of expression in the healthy oocyte and blastocyst is presented, including the identification of oocyte-unique and blastocyst-unique genes. The microarray data from this study will guide experiments to identify cases where normal gene expression is disrupted.

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