A team of researchers from Texas A&M University, the University of Pennsylvania and the Children’s Hospital of Philadelphia have uncovered genetic variants associated with sleep regulation. The work, published in Science Advances, is the first to use “variant-to-gene” mapping to identify genes and pathways associated with sleep.
GWAS – a tired approach?
Sleep is arguably one of the most important aspects of human life. Disrupted or low-quality rest can be debilitating, yet our understanding of the biology of sleep regulation is lacking. Multiple studies have identified genetic variants associated with insomnia, yet most have failed to validate these findings. This is partly because variants uncovered in a genome-wide association study (GWAS) are often found in regulatory regions, far from the closest neighbouring gene. This can make it difficult to identify the specific genes that impact any trait or disease. It is therefore vital to understand the functional effects any variants have on the genome.
In an effort to address this issue and understand more about how genetic variants influence sleep regulation, Alex Keene and his team used a “variant-to-gene” mapping approach to predict the impact of variants associated with insomnia. The approach combined previously obtained GWAS data with ATAC-Seq/promoter-focused Capture C data to predict genes that are implicated in insomnia-related phenotypes. The experimental approach used in the study is described in Figure 1.
Figure 1: Image describing the experimental approach used by the team. GWAS data and ATAC-Seq/promoter-focused Capture C data were combined, before candidate genes were taking forward for screening in vivo. Adapted from Palermo et al. 2023.
(RNA)-Interfering with sleep
This approach uncovered 88 candidate genes that were associated with insomnia-related phenotypes. However, this data alone was still not enough to explain the genetics underpinning sleep regulation. The team therefore moved into an in vivo model of disease, using the model organism drosophila melanogaster.
The researchers used RNA-interference to knock-down expression of relevant candidate genes in the fruit fly models. This uncovered associations with “short-sleeping” and “long-sleeping” phenotypes – in other words, flies that slept for shorter or longer durations. For example, short-sleeping was observed in flies that had reduced expression of the homothorax gene – the human ortholog of which has been previously implicated in restless leg syndrome and sleep disruption.
Of particular interest was a long-sleeping phenotype associated with knock-down of the PIG-Q gene, a crucial component of the GPI-anchoring biosynthesis pathway. GPI-anchoring is a common protein modification vital for cellular integrity and the associated proteins are highly conserved across species. Given this information, the team chose to further explore this gene’s role in sleep regulation.
Despite their invaluable role as a model organism, fruit flies do not have as much in common with humans as certain other organisms. In light of this, the next stages of the experiment focused on the role of PIG-Q in zebrafish. PIG-Q expression was knocked-out using a CRISPR-Cas9 system, resulting in a long-sleeping phenotype in the fish. This validated the findings that PIG-Q is a sleep regulator, conserved across different species.
Don’t sleep on it
Whilst often overlooked, sleep conditions such as insomnia and narcolepsy are life-altering conditions, and even short-term sleep disruption can have detrimental, system-wide effects on the body. This highlights how crucial it is to get to the bottom of the biological mechanisms at play.
The team’s next steps are to investigate the role of GPI-anchor protein modifications in more depth, to find out what specifically causes the long-sleeping phenotype. An understanding of this link to sleep disruption could be vital in developing new therapies for these illnesses. Not only that, the study has validated the usefulness of the variant-to-gene approach and the team are committed to continued use of the method. Discussing what makes the approach so impactful, Keene praised his colleagues. He stated: “we each bring a different area of expertise that allowed for this collaboration’s ultimate effectiveness.”