A trail of crumbs and pebbles
Authors:   , Timpanaro Ilia Sarah 
Published: September 2024

Genetics is traditionally associated with the study of protein-coding regions of DNA, or genes. However, much of the human genome does not encode proteins but instead has a regulatory function. These regulatory sequences, known as regulatory elements, modulate gene expression with spatial and temporal precision, playing a critical role in cell differentiation, development, and disease. This thesis investigates the role of regulatory elements in neurodegenerative diseases, focusing on how sequence variants, referred to as regulatory variants, can alter regulatory function and contribute to disease. While regulatory variants are not often investigated in the context of disease, they may account for the unknown genetic causes of many disorders.
Investigating regulatory variants is inherently challenging. In contrast to protein-coding genes, where mutations lead to relatively predictable effects on proteins, regulatory elements do not follow such straightforward rules. Chapter 2 introduces a novel approach to functionally characterize regulatory variants in the context of Parkinson’s disease (PD), focusing on a regulatory element influencing a gene implicated in the disease, SNCA. PD is a progressive neurodegenerative disorder characterized by motor impairment and cognitive decline. Using human cultured cells, we introduced sequence variations in the chosen regulatory element and assessed their functional effects. Employing precise genome-editing techniques, we minimized off-target effects, enabling reliable conclusions. Our results demonstrated that two of the variants significantly affected SNCA expression, potentially influencing PD risk.
In Chapter 3, the focus shifts to Amyotrophic Lateral Sclerosis (ALS), a fatal neurodegenerative disease characterized by the degeneration of motor neurons, leading to paralysis and respiratory failure. ALS has a complex genetic architecture, with many cases linked to rare variants. Identifying rare variants is difficult because standard methods, such as genome-wide association studies, are optimized for common variants. To address this, we developed a computational method based on rare variants association analysis to identify rare variants in regulatory elements of the motor cortex, a brain region severely affected in ALS. Our analysis revealed a regulatory element with potential protective effects. Functional experiments in ALS patient-derived motor neurons showed that downregulation of UBR5, a gene that is likely regulated by this element, alleviated disease phenotypes. Though the exact role of the individual mutations remains to be elucidated, these results suggest that variants may reduce UBR5 expression and thus confer protection against ALS, highlighting the role of UBR5 and its regulatory element as potential genetic modifiers.
Chapter 4 focuses on the systematic discovery of regulatory elements in the spinal cord, the primary site of degeneration in ALS. Using human post-mortem tissue, we applied several experimental techniques to map regulatory elements. Despite technical limitations due to DNA degradation in post-mortem samples, we successfully identified regulatory elements specific to spinal cord cell types. This allowed us to generate the first comprehensive single cell regulatory dataset for the human spinal cord, which can be used to identify disease-associated regulatory variants in future research.
This thesis underscores the critical role of regulatory elements in neurodegenerative diseases, providing new insights into the genetic mechanisms underlying PD and ALS.