title: The role of non-coding tandem repeat DNA and non-LTR retrotransposons in Amyotrophic Lateral Sclerosis risk loci
Creators: name: Marshall, Jack
Publisher: name: University of Liverpool Repository
Year: 2021

description: Genome-wide association studies and functional data have shown that there is a genetic basis contributing to sporadic and familial forms of amyotrophic lateral sclerosis (ALS), with a vast plethora of genes being associated with the disease. Since the discovery of the C9orf72 intronic repeat expansion there has been a growing awareness of non-coding repetitive DNA being associated with ALS risk. Originally labelled “junk DNA”, non-coding repetitive DNA is now known to be vital in regulating and shaping the human genome and has become of particular interest in the context of disease risk and pathogenesis. Repetitive DNA can be found in two forms: static and mobile, both of which were addressed in this PhD project, with an emphasis on variable number tandem repeats (VNTRs) and non-long terminal repeat retrotransposons. Previous studies have demonstrated that polymorphic VNTRs can be associated with predisposition to disease and this often correlated with the differential transcriptional regulatory properties of the VNTR based upon the copy number of the repeat element. Analysis of the ALS risk gene CFAP410 led to the identification of an intronic VNTR, with the discovery and characterisation of novel genetic variants present only in ALS patients. This VNTR was also shown to be functional in two reporter gene assays: both driving expression in the absence of a minimal promoter and also acting as a transcriptional regulatory domain, modulating expression in an allelic dependent manner on the basis of repeat copy number. Loss of function variants of NEK1 have been found to be associated with ALS risk. Current studies have focused on the coding regions of this locus, therefore our work aimed to address non-coding variation at this region, with the hypothesis that such genetic variation could impact NEK1 gene regulation. This analysis led to the discovery of an intronic SINE-VNTR-Alu (SVA) retrotransposon within NEK1, which was found to be polymorphic within three domains: the 5’ CT element, central VNTR and 3’ poly A tail. Of note, two CT element variants were only found in ALS patients from a UK MNDA cohort. This region was then assessed using Isaac variant caller, facilitating high-throughput characterisation of SVA genetic variation within cohorts of Project MinE, which still agreed with the previous trend in genotype frequency but one particular cohort did not conform to that observed in the UK MNDA cohort. The NEK1 SVA was tested functionally and demonstrated regulatory function in vitro, inducing significant repression within two reporter gene constructs, inducing both cell line and orientation specific expression profiles. By utilising the CRISPR Cas 9 system it was possible to excise the SVA element in HEK293 cells, but under basal conditions this deletion did not induce a significant change in NEK1 gene expression. Ultimately this work aimed to raise the profile of VNTRs and SVA elements as potential sources of missing heritability in complex disease and to better understand their function in gene regulation.