Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disorder of the tyrosine
degradation pathway. The defective fumarylacetoacetate hydrolase enzyme causes the
accumulation of upstream metabolites such as fumarylacetoacetate (FAA), maleylacetoacetate
(MAA), succinylacetone (SA) and p-hydroxyphenylpyruvic acid (pHPPA). In vitro and in vivo
studies showed that the accumulation of these metabolites are detrimental to cell homeostasis, by
inducing cell cycle arrest, apoptosis, and endoplasmic reticulum stress, depleting GSH, inhibiting
DNA ligase, causing chromosomal instability, etc. For in vivo studies different models of HT1 were
developed. Most notably was the fah deficient mouse, whose neonatally lethal phenotype is
rescued by the administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC).
Although, this model most closely resembles the human phenotype with elevated tyrosine levels
and the development of hepatocellular carcinoma (HCC), the model is not human genome based.
Both the in vitro and in vivo studies suggested that DNA repair is affected in HT1.
However, it is not yet clear which DNA repair mechanisms are affected and if only protein
functionality is affected, or if expression of DNA repair proteins are also affected.
Characteristic of HT1 is the high prevalence of HCC and the presence of liver mosaicism.
The liver mosaicism observed in HT1 patients are the result of reversion of the inherited mutation
to wild-type. The general consensus is that the reversion is the result of a true back mutation.
However, the mechanism underlying the back mutation is still unresolved.
It was suggested that cancer develops either through a chromosomal instability mutator
phenotype, a microsatellite instability mutator phenotype, or a point mutation instability mutator
phenotype. In HT1 only chromosomal instability was reported.
The aims of this study were to contribute to the understanding of the molecular basis of the
genetic mosaicism in hereditary tyrosinemia type 1. More specifically, determine whether baseand
nucleotide DNA repair mechanisms are affected and to what extent, and to determine if
microsatellite instability is found in HT1. To achieve these aims, a parallel approach was followed:
i.e. to develop a HT1 hepatic cell model and to use HT1 related models and HT1 patient material.
To assess the molecular basis of the genetic mosaicism in HT1, the comet assay, gene expression
assays, microsatellite instability assays, high resolution melting and dideoxy sequencing
techniques were employed. Results from the comet assay showed that the HT1 accumulating metabolites, SA and
pHPPA, decreased the capacity of cells for base- and nucleotide excision repair. Gene expression
assays showed that short term exposure to SA and/or pHPPA do not affect expression of hOGG1
or ERCC1. The expression of these genes were, however, low in HT1 patient samples.
Microsatellite instability assays showed allelic imbalance on chromosome 7 of the mouse genome,
and microsatellite instability in the lymphocytes of HT1 patients. Although high resolution melt and
sequencing results did not reveal any de novo mutations in fah or hprt1, the appearance of de
novo mutations on other parts of the genome can not be ruled out.
To conclude, results presented in this thesis, for the first time show that in HT1 the initiating
proteins of the base- and nucleotide repair mechanisms are affected, the gene expression of DNA
repair proteins are low, and microsatellite instability is found in HT1. By contributing to the
elucidation of the mechanism underlying the development of HT1-associated HCC, and providing
evidence for the development of a mutator phenotype, the results presented in this thesis
contributes to the understanding of the molecular mechanisms underlying the genetic mosaicism in
HT1. In addition to these contributions, a hypothesis is posited, which suggests that a point
mutation instability (PIN) mutator phenotype is the mechanism underlying the mutation reversions
seen in HT1. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2012
| Identifer | oai:union.ndltd.org:NWUBOLOKA1/oai:dspace.nwu.ac.za:10394/8517 |
| Date | January 2011 |
| Creators | Van Dyk, Etresia |
| Publisher | North-West University |
| Source Sets | North-West University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
Page generated in 0.002 seconds