High-Throughput Testing Reveals Genetic Programs, Pathogenic Variants in Neurodegenerative Disease30.08.2022
Researchers at the University of California, Los Angeles have identified noncoding genetic regulatory elements that impact the risk of dementia from Alzheimer's disease and the pathologically related progressive supranuclear palsy (PSP).
In a study published yesterday in the journal Science, the researchers integrated a massively parallel multiplexed reporter assay and a pooled CRISPR screen to identify and characterize noncoding disease-associated genetic variants in a single experiment. The group calls this strategy a roadmap for conducting further such experiments in the future.
Daniel Geschwind, a professor of neurology, psychiatry, and genetics at UCLA and the study's corresponding author, explained that his group's method helps account for certain shortcomings in genome-wide association studies. While GWAS have revolutionized the detection of genetic loci, they cannot identify causal variants and the genes they impact.
"This poses an enormous challenge," he said, "because most common susceptibility loci reside in noncoding genomic regions and are composed of many correlated polymorphisms owing to linkage disequilibrium. We reasoned that we could use newly developed high-throughput approaches that allow us to measure the effects of genetic variants on transcription to bridge this critical gap in the field."
Genetic variants associated with Alzheimer's and PSP, for instance, have proven challenging to study, as many disease-associated loci overlap noncoding regions and contain hundreds of variants subject to linkage disequilibrium, which complicates the identification of regulatory variants.
Further complicating things, computational methods for predicting disease risk-associated variants have limited utility when assessing noncoding regions.
To overcome these limitations, Geschwind and his colleagues barcoded both alleles of 5,706 genetic variants in 25 Alzheimer’s-associated loci and nine PSP-associated loci and cloned them into an expression library, on which they conducted a massively parallel reporter assay.
From this, they identified 320 functional variants, 42 of which were deemed "high confidence" based on their functional annotations and their locations within regulatory regions known to be active in brain tissue.
The researchers validated these 42 functional variants in a pooled CRISPR screen using induced pluripotent stem cells (iPSCs) and CROP-seq, a method for benefiting from both pooled and arrayed screens by combining CRISPR screening and single-cell transcriptome sequencing.
"This allows us … to move from thousands of genetic variants associated with a disease to identifying which are functional and which genes they impact," Geschwind said in a statement associated with the study.
Their screen identified several new candidate risk genes for both Alzheimer's and PSP, which the investigators tested experimentally through targeted CRISPRi assays.
Results showed at least one PSP-related pathogenic mechanism, in which multiple disease-associated loci acted additively to disrupt a core set of transcription factors, whose dysregulation is consistent with risk predictions for PSP.
Geschwind said that this finding suggests that targeting a network of genes, as opposed to a single pathogenic variant, could become an effective therapeutic approach.
"We’re entering in a new stage of therapies — it’s beginning to be plausible to think about targeting networks," he said.
Geschwind cautioned that the success of the current study "does not mean that we can jettison the kind of detailed, careful experimentation studying individual genes in model systems."
Rather, he explained, his group's method provides a key experimental step between GWAS and the understanding of disease mechanisms.
Following this publication, Geschwind said, "we are expanding this study to assess the role of rare variants in addition to common variants."