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Título: Transmission and distribution pattern of disease-related genes in early mammalian embryos
Autores: Dean-Booth, Nicola.
Fecha: 2006
Publicador: McGill University - MCGILL
Fuente:
Tipo: Electronic Thesis or Dissertation
Tema: Biology, Genetics.
Descripción: Knowledge of the mechanisms involved in the transmission of pathogenic mutations during early development is paramount to understanding the inheritance of human disease. The current thesis investigated the transmission to early embryos of the (CTG)n repeat in the human dystrophia myotonica-protein kinase (DMPK) gene, which is expanded in dystrophia myotonica type 1 (DM1) and of heteroplasmic mitochondrial DNA (mtDNA). For each of these studies, single in-vitro cultured human or in-vivo generated mouse oocytes and embryos were genotyped for the specific DNA sequences of interest.
Initially, genotyping was optimised at the single-cell level using different protocols and methods of product detection. Fluorescent-based PCR analysis of the mutation of interest multiplexed with a linked polymorphic marker was found to provide maximal efficiency and accuracy. Once standardised, these tests were applied to clinical preimplantation genetic diagnosis (PGD) and served as the experimental basis for further work presented in this thesis. This included two studies investigating the transmission of the DMPK repeat. In the first study, the objective was to determine whether transmission ratio distortion (TRD) in favour of larger normal-sized alleles ((CTG)19-37) was evident during early human embryogenesis. A statistically significant TRD in favour of DMPK (CTG)19-37 repeats was observed in embryos, which remained significant when female embryos were considered separately. This TRD was shown to be specifically due to the transmission of repeats in the (CTG)19-37 range. The developmental time-point of TRD of these alleles was suggested to occur around the time of fertilisation. In addition, instability in this repeat length was demonstrated during paternal transmission. The second objective was to further elucidate the timing and variability of the intergenerational enlargements observed in expanded DMPK alleles in early development. From our analysis, repeat expansion was found to occur in oogenesis, most likely before the completion of meiosis I. Furthermore, variable degrees of expansion were observed in each cohort of embryos, which are comparable to the phenotypic variability seen between DM1 siblings.
In the final chapter, the segregation pattern of murine heteroplasmic mtDNA in oocytes and early embryos was examined in order to determine the feasibility of PGD for mtDNA mutations. This study demonstrated that the mean level of heteroplasmy in the polar body of an oocyte or a single blastomere of an embryo was representative of the level in the embryo as a whole and, therefore, PGD would be a viable option for women heteroplasmic for mtDNA point mutations.
Idioma: en