William Gibson

To our knowledge age-related loss of chromosome Y (LOY) in circulating leukocytes is the most common somatic genetic aberration. Many recent epidemiology studies have found robust associations between LOY in leukocytes and age-related diseases such as blood and solid tumour cancers, Alzheimer’s disease, and macular degeneration. Despite these associations, the prevalence and mechanisms of LOY in non-hematopoietic cell-types are not well characterized. In response, the need for bioinformatic methods to analyse Y chromosome ploidy across multiple genomic/transcriptomic datatypes has escalated. In the past, the Y chromosome was commonly removed from genomic analyses for several reasons including low gene count, haploidy, lack of biological interest and short-read mapping difficulties. Resultingly, methods for investigating chromosome Y specific trends using next-generation sequencing have suffered and require improvement. The main objective of this thesis was two-fold. First, to improve methods of Y chromosome aneuploidy detection using whole genome sequencing and single-nuclei RNA sequencing. Second, to use these improved methods to provide estimates of loss of Y (LOY) in brain tissue – which had not previously been established in humans. Using genomic characteristics such as mappability, GC content, and read alignment filtering I was able to improve LOY detection in both WGS and single-nuclei RNA-seq. Given high sequence similarity between the X and Y chromosome, strict mappability filtering improves, and smooths read depth estimates of Y chromosome aneuploidy. Using these methods we estimate that of the elderly male population represented in this cohort (median age = 87.5), LOY was found in 13.8% (11/123) of blood samples, 0% (0/159) in prefrontal cortex and 0% (0/78) cerebellum samples. Despite this, we found a significant association between age and reduced Y ploidy in the dorsolateral prefrontal cortex (R=-0.35, p=3.9x10-⁵), suggesting low-frequency LOY may be occurring in the cortex. In single-nuclei data from the dorsolateral prefrontal cortex we found 8.6% of cells lacked a Y chromosome. LOY was enriched in the glial cells, and particularly the microglia where 33% of male cells were affected. Although further evidence is required, LOY within the frontal cortex (and specifically the microglia) may represent an understudied factor in cognitive decline and neurodegeneration.

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Intracranial aneurysms (IA), a common disease that occurs when cerebral arteries weaken and expand, can lead to subarachnoid hemorrhage upon rupture. The prevalence of IA is estimated to be around 3% and is known to increase with age. A small subset of the patient population has a familial form IA, where two or more first- to third- degree relatives have IA. At this time, one gene, THSD1, has been associated with familial IA (FIA). Here we present the preliminary findings from whole exome sequencing on five families diagnosed with FIA. Each family appears to have Mendelian segregation of disease (autosomal dominant, autosomal recessive, or X-linked) and has had their aneurysms clinically confirmed through brain imaging. Sequencing data from the proband of each family was used to identify family-specific candidate genes and was overlapped between families to identify genes that contain rare, possibly pathogenic variants in three or more families. Four genes -- DST, CRIPAK, DNAH1, and TTN --were found to contain rare variants in four out of the five families. Three top candidate genes were selected based on gene function or previous association to cerebral vascular disease from 38 genes that contain rare variants in three out of the five families.

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The discovery of leptin and other humoral signals which regulate food intake and energy expenditure has greatly contributed to our understanding of molecular pathways controlling energy homeostasis. Leptin produced by adipocytes, insulin produced by the pancreas, and ghrelin produced by the stomach all contribute to the body’s energy balance. One question remaining is whether the lipid transport system also plays a role.Our hypothesis is that lipid clearance is important in the maintenance of energy homeostasis. The low-density lipoprotein receptor (Ldlr) is a key molecule involved with lipid clearance. The experiments presented in this thesis used the Ldlr-/- mouse to study the Ldlr’s role in energy balance. One aim of this thesis was to provide a detailed analysis of the energy balance phenotype of the Ldlr-/- mouse. Another aim of this thesis was to use the Ldlr-/- mouse to study the potential interaction between Ldlr and the leptin signaling pathway.Adult Ldlr-/- mice and Ldlr+/+ controls on a C57BL/6J background were fed either a chow or a high-fat, high-sucrose Western-type diet (WTD) for eight weeks. Physiological studies of food intake, energy expenditure, activity, heat production, insulin sensitivity, and leptin responsiveness were performed. As well, the effect of these diet interventions on circulating leptin and on leptin gene expression was examined.On the chow diet, Ldlr-/- mice had lower energy expenditure and higher activity levels relative to controls. On the WTD, Ldlr-/- mice gained less weight relative to Ldlr+/+ mice, specifically gaining less fat mass. Increased thermogenesis in Ldlr-/- mice fed the WTD was detected. Additionally, leptin responsiveness was blunted in chow-fed Ldlr-/- mice, suggesting a novel role for the Ldlr pathway that extends to leptin’s regulation of energy balance.In addition to its known role in lipid transport, these results from the Ldlr-/- mouse demonstrate the importance of the Ldlr in regulating energy homeostasis and suggest a direct physiological link between dyslipidemia and energy balance.

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