Ging, Heather; (2020) Investigating the Role of Modifiers in Trinucleotide Repeat Diseases. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Friedreich’s Ataxia (FRDA) and Huntington’s disease (HD) are trinucleotide repeat diseases, resulting from homozygous expanded GAA and heterozygous expanded CAG repeats, respectively. The pathogenic repeat length is inversely correlated to disease onset and severity; the longer the repeat, the earlier the onset and the more severe the phenotype. However, repeat size does not fully explain the phenotypic variability. This report investigates if repeat interruptions act as disease modifiers. Small GAA repeat interruptions were common in the FRDA cohort in contrast to large interruptions, yet we did not expose the base pair configuration. Clone sequencing of the CAG repeat revealed that the age at onset is more accurately predicted based on the length of the pure CAG repeat. The penultimate synonymous CAA interruption was determined to modify onset with its loss hastening onset and an additional CAA interruption delaying onset. These results have been substantiated by recent reports (GEM-HD, 2019; Wright et al., 2019). Complementing clone sequencing, the base pair configuration of the HD pathogenic region was determined using next- and third-generation sequencing technologies, revealing that Illumina MiSeq sequencing was most applicable to our samples based on DNA quantity and quality. In contrast, Pacific Biosciences single molecule real time sequencing and Oxford Nanopore sequencing were limited by sample concentration. A prominent characteristic of HD is somatic mosaicism, which mirrors the specific neurodegeneration. To understand the contribution of CAG repeat instability to HD pathogenesis, the somatic mosaicism profile in six HD post-mortem brains was analysed by Illumina MiSeq, which determines the proportion of common variants (small CAG repeat changes) and SP-PCR, which quantifies large CAG repeat changes. Illumina MiSeq revealed that the striatum contained the highest level of instability. In contrast, SP-PCR determined that the cortical regions displayed the greatest levels of instability. These results complement the somatic mosaicism profiles previously determined in Kennedy et al., 2003, and supports the hypothesis that cells with the largest CAG repeat sizes are primarily lost. Emerging evidence highlights DNA repair pathway genes as modifiers of CAG repeat instability and HD phenotype (GEM-HD, 2019). In our HD cohort, who were genotyped for the implicated disease modifying DNA repair pathway SNPs, we similarly show that some of the phenotypic variability can be attributed to these genetic variants, specifically in FAN1. The future use of more physiological disease models, such as induced pluripotent stem cells, will aid in deciphering the exact role of DNA repair pathway genes as modifiers of instability and thus, disease progression.

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