Abstract
Parkinson's Disease (PD) is a globally increasing neurodegenerative disorder, posing growing economic, societal, and personal burdens. Genetic and environmental factors contribute to PD development, with glucocerebrosidase (GBA1) gene variants emerging as significant risk factors. Initially thought to be benign, GBA1 variations are now known to substantially elevate PD risk and worsen disease progression.
This research aims to enhance our understanding of GBA1 variants' impact on PD by leveraging Oxford Nanopore Technologies (ONT) sequencing, enabling in-depth analysis of this complex genetic landscape. This study builds upon a previously established GBA1 nanopore sequencing method, refined for use in PD patient samples from the clinically diagnosed NZP3 PD cohort. Sequencing 96 samples simultaneously, we achieved high read-depth (>2000x) across an 8.9kb GBA1 amplicon, detecting 24 PD-associated GBA1 variants, as well as variants of unclear significance. Notably, PD patients exhibited a higher variant count and an increased prevalence of dementia compared to controls. Molecular haplotyping revealed 55 unique haplotypes, with common haplotypes linked to earlier PD onset and diagnosis age, emphasizing the role of common variants on PD progression.
Expanding beyond PCR-dependent methods, I explored two novel nanopore sequencing techniques: CRISPR/Cas9-based genomic cuts and adaptive sampling. These methods offer the potential to identify structural variations, methylation, and genes beyond GBA1 more directly. The CRISPR/Cas9 approach successfully targeted and sequenced GBA1, while adaptive sampling enabled the selective sequencing of an 803-gene neurological panel, albeit with sensitivity and precision limitations.
The study underscores the importance of common GBA1 variation in PD development and progression. Future research should prioritise investigating such common variation, complementing the focus on coding variants and familial risk factors. As most sporadic PD cases lack known genetic risk factors, understanding nuanced variation is crucial for predicting disease onset and progression.
Preliminary research on variation in GBA1 (and other genes) may uncover additional risk factors and potential therapeutic targets for PD. Future PD treatments must target early intervention and pre-symptomatic individuals, in contrast to current symptom-focused therapies. Genetic screening plays a pivotal role in identifying individuals at risk, facilitating drug trials, and tailoring interventions to genetic risk factors, both common and rare, influencing disease outcomes and drug effectiveness.