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Genetic and Environmental Modifiers of Somatic Trinucleotide Repeat Dynamics University of Glasgow Institute of Biomedical and Life Sciences Division of Molecular Genetics Genetic and Environmental Modifiers of Somatic Trinucleotide Repeat Dynamics Mario Gomes-Pereira Thesis submitted for the degree of Doctor of Philosophy June 2002 ProQuest Number: 13833915 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 13833915 Published by ProQuest LLC(2019). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 GLASGOW UNIVERSITY lUBRARY: Abstract The expansion of CAG'CTG trinucleotide repeat sequences has been identified as the genetic cause of several human diseases, including myotonic dystrophy type 1, Huntington disease, and an ever-increasing number of spinocerebellar ataxias. Once above a size threshold, the repeats become dramatically unstable in the germline and also throughout the soma, with a marked bias towards further expansion. Such expansions constitute a unique form of dynamic mutation, whose mechanism is poorly understood. While germline instability serves as the molecular basis for genetic anticipation; age-dependent, tissue-specific somatic instability most likely contributes to the tissue specificity, phenotypic variability and progressive nature of these conditions. The study of the mutation mechanism is therefore of major interest, as it may provide valuable clues towards a better understanding of disease pathophysiology. It is generally assumed that the repeat length changes arise through DNA polymerase slippage during DNA replication, however no direct evidence exists to support this hypothesis in mammalian systems. Transgenic mouse models of unstable CAG'CTG repeats have been previously generated, and shown to recreate the dynamic nature of somatic mosaicism observed in humans. Tissues from these mice have now been used in order to establish an in vitro cell culture system, where the repeat dynamics could be investigated under controlled conditions. Monitoring of repeat stability in these cells over long periods of time, and numerous population doublings, has revealed the progressive accumulation of larger alleles, as a result of repeat length changes in vitro, confirmed by single cell cloning. Selection of cells carrying longer repeats was observed during the first few passages of the cultures, and frequent additional selective sweeps were also detected at later stages. The highest levels of instability were observed in cultured kidney cells, whilst the transgene remained relatively stable in eye cells and very stable in lung cells, paralleling previous in vivo observations. More importantly, the levels of repeat instability in cultured cells did not correlate with cell proliferation rates, rejecting a simple association between length change mutations and cell division, and suggesting an important role for additional cell type-specific and possibly environmental trans-acting modifiers of repeat metabolism. The effects of multiple genotoxic agents on the mutational dynamics of expanded trinucleotide repeats were assessed in this tissue culture model of unstable DNA. The drugs tested were selected based on their ability to affect cell cycle progression, DNA polymerase activity, DNA methylation, intracellular levels of oxidative stress or DNA conformational metabolism. The analysis led to the identification of chemicals, such as aspirin, 5-azacytidine and 1-B-D- arabinofuranosyl-cytosine that resulted in the deceleration of the rate of trinucleotide repeat expansion, particularly in a kidney clonal cell line carrying rapidly expanding repetitive tracts. These observations were reported in the absence of major changes in the rates of cell turnover. In contrast, forced cell cycle progression by exposure to caffeine resulted in a significantly higher rate of triplet repeat expansion. Increased levels of oxidative stress, generated in culture by exposure to 2 a variety of drugs, were associated with reduced levels of repeat size variability, most likely through means of cell selection in culture. Since pathology in CAG#CTG-associated diseases is mediated by a variety of complex and unrelated molecular pathways, drug induced modification of DNA dynamics could present a possible therapeutical route for these disorders. Specifically, chemical treatments that resulted in suppression of somatic repeat expansion would be expected to be beneficial, whilst reversion of the expanded mutant repeat to the normal repeat size range, observed in the general population, would be predicted to be curative. Although preliminary, the findings described in this study may open new avenues in the search for novel therapeutical strategies. Mechanistic models of repeat length mutation based on DNA replication, recombination and repair have been proposed. The latter have implied the involvement of mammalian M utS homologues ( M sh2 , Msh3 and M sh 6 ). In order to gain further insight into the molecular mechanisms driving trinucleotide repeat mutation, the involvement of a mammalian M utL homologue ( Pms2) in the mutation dynamics was investigated. No significant differences were observed between Pms2+I+ and P m s2+I' mice, suggesting that a single functional Pm s2 allele is sufficient to maintain high levels of somatic mosaicism. The levels of Pms2 mRNA and protein in heterozygotes deficient for Pms2 have not yet been investigated. In contrast to what would be predicted by the replication slippage model, lower levels of trinucleotide somatic mosaicism were detected in homozygous Pms2-nu\l mice, compared with age-matched controls, carrying either one or two functional copies of the Pm s2 allele. In addition, a higher frequency of rare but large deletion events was detected in Pms2'1' animals. Both results proved statistically significant by single molecule analysis. These findings imply that, not only MMR enzymes that directly bind to DNA, but also proteins that are subsequently recruited by MutS proteins, play a central role in the accumulation of repeat length changes, arguing against a mutation mechanism mediated by stabilisation of alternative DNA secondary structures by MMR proteins. MMR gene polymorphisms and variants might therefore be considered potential determinants of trinucleotide repeat instability in humans, predicted to affect both age of onset and disease progression. 3 Let us learn to dream.... then perhaps we shall find the truth. Friedrich August Kekule Tambem eu, tambem eu, joguei as escondidas, fiz baloiqos, tive bolas, berlindes, papagaios, automdveis de cor da, cavalinhos... Depois cresci, tornei-me do tamanho que hoje tenho, os brinquedos perdi-os, os meus bibes deixaram de servir-me. Mas nem tudo sefoi: ficou-me, dos tempos de menino, esta alegria ingenua perante as coisas novas e esta vontade de brincar. Vida! nao me venhas roubar o meu tesouro: nao te importes que eu ria, que eu salte como dantes. E se eu riscar os muros ou quebrar algum vidro ralha, ralha comigo, mas de manso... Eu tinha um bibe azul... Tinha berlindes, tinha bolas, cavalos, papagaios... A minha Mae ralhava assim como quern beija... E quantas vezes eu, so pra ouvi-la ralhar, parti os vidros da janela e desenhei bonecos naparede... Vida!, ralha tambem, ralha, se eu tefizer maldades, mas de manso, como se fosse ainda a minha Mae... Sebastiao da Gama 4 Table of contents Abstract ..............................................................................................................................................................2 Table of contents................................................................................................................................................5 List of tables .................................................................................................................................................... 10 List of figures ................................................................................................................................................... 11 Acknowledgements ..........................................................................................................................................14 List of Abbreviations .......................................................................................................................................18 1. Introduction .............................................................................................................................................. 21 1.1. Trinucleotide repeats and human disease........................................................................................... 21 1.2. Molecular pathogenesis......................................................................................................................24 1.2.1. Polyglutamine diseases ........................................................................................................... 24 1.2.1.1. Toxicity of polyglutamine tracts ................................................................................ 25 1.2.1.2. Poly glutamine protein intranuclear inclusions ...........................................................26
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