Preimplantation diagnosis of X-linked disease
Author(s)
Kontogianni., Eleni
Type
Thesis
Abstract
Preimplantation diagnosis of inherited disease following in vitro fertilization (IVF) allows the transfer of unaffected embryos to the uterus avoiding the termination of an affected pregnancy in couples at risk of transmitting an inherited disorder. Possible approaches for X-linked recessive disease include transfer of females by identification of embryo sex, biochemical assay of enzyme deficiencies or specific detection of the mutation. DNA amplification using the polymerase chain reaction (PCR) of Y- specific repeats from single blastomeres biopsied at cleavage stages identified male embryos. Following transfer of females, the first pregnancies were established. The sex of the remaining embryos was confirmed by PCR. Examination of single blastomeres from disaggregated embryos demonstrated that amplification failure occurs in a minority. Co-amplification of X- and Y-specific repeats from single cells was possible. Different lysis protocols were compared and a two-step method shown to minimise failure. A minimum of two cells is nevertheless necessary. Detection of enzyme deficiencies by biochemical assay requires that embryo-derived enzyme predominates over maternal. To examine embryonic gene expression, qualitative and quantitative aspects of protein synthesis were studied. Protein synthesis was constant during cleavage, when most qualitative changes occurred, and increased significantly at the blastocyst stage. Transcriptional inhibition had no effect on rate of synthesis at early stages but blocked the rise in blastocysts suggesting persistence of maternal mRNA after activation of the embryonic genome. Lesch-Nyhan syndrome is caused by deficiency of X-linked hypoxanthine phosphoribosyl transferase (HPRT). HPRT activity was dependent on transcription and was different in males and females at the blastocyst stage, suggesting that embryo coded enzyme predominates over the maternal. However, diagnosis based on the product of gene expression requires further work. Diagnosis of the predominant mutations causing single gene defects isnow possible by PCR. However, many X-linked defects are heterogeneous.
Version
Open Access
Date Awarded
1994
Publisher Department
Department of Medicine
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)