Flower, Michael; (2019) Genetic variation in DNA repair proteins modifies the course of Huntington’s disease. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Huntington’s disease (HD) is caused by a CAG repeat expansion in HTT on chromosome 4. Onset and progression are inversely correlated with repeat length, but a significant proportion of the variability in each is due to modifiers elsewhere in the genome. Recent genome-wide association studies have identified the DNA repair genes FAN1 and MSH3 as modifiers of onset and progression respectively. This thesis finds that variants associated with HD disease course also influence onset in other polyglutamine diseases, suggesting a shared pathogenic mechanism involving DNA repair. In blood from HD patients there is significant transcriptional dysregulation, particularly involving immune, metabolic and DNA repair pathways, which correlates with disease severity, parallels dysregulation seen in the most affected HD brain regions and overlaps with Alzheimer’s disease. To study the role of DNA repair, several cell models of somatic instability were developed, including patient-derived lymphoblasts and induced pluripotent stem cells, which show exponential repeat expansion that continues in differentiated medium spiny neurons (MSNs). In a FAN1 knockout U20S cell model of HD, FAN1 is shown to protect against repeat instability, and this function is dependent on protein concentration and CAG repeat length, but does not require its nuclease activity. shRNA-mediated FAN1 knockdown accelerates repeat expansion in both patient-derived iPSCs and MSNs. Through chromatin immunoprecipitation, FAN1 is shown to bind, but not specifically target, CAG repeat DNA. AAV9-mediated miRNA Fan1 knockdown in the striatum and liver of R6/2 mice did not accelerate repeat expansion, likely because only 23% knockdown was achieved. Illumina sequencing of the MSH3 region that influences HD progression identified a repeat variant that is associated with decreased MSH3 expression, reduced somatic expansion, delayed onset and slower progression in HD and myotonic dystrophy type 1 (DM1). These results suggest MSH3 promotes and FAN1 protects against repeat instability, which in turn influences the course of repeat expansion diseases.

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