Biology subjects

Pack, A. I.

Publications and source records attributed to Pack, A. I..

10 recordsLinked to original sources

bioRxiv2026

Cell-specific variant-to-gene mapping identifies conserved neural and glial regulators of sleep

Excessive daytime sleepiness (EDS) is a heterogeneous phenotype with little known of its genetic basis. Large-scale genome-wide association studies (GWAS) have reported genomic loci associated with EDS, though since most of these are non-coding, the causal gene(s) underlying the association are not known. Additionally, the cell types in which these genes exert their effects on sleep have not been functionally explored in vivo. Here, we employed a chromatin-based variant-to-gene mapping approach to first implicate candidate effector genes at EDS GWAS loci in human-derived neural and glial cell lines. Subsequent cell type-specific RNAi knockdown of orthologous genes using neural and glial GAL4 drivers in Drosophila confirmed cell-specific regulation of sleep by these GWAS-implicated effector genes. Among these, ruby (ortholog to AP3B2), a component of the AP-3 vesicular trafficking complex emerged as a robust sleep regulator. Targeted knockdown in flies localized ruby function to astrocyte-like glia, where loss of ruby increased sleep duration. The conserved role of ruby/ ap3b2 was validated in zebrafish where CRISPR-mediated loss increased daytime sleep. Together, these findings show that physical variant-to-gene mapping predicted cell-type-specific gene function for complex sleep traits and revealed ruby/AP3B2 as a conserved glial regulator of sleep and arousal. This work provides a generalizable framework for connecting non-coding GWAS variants and their corresponding effector genes to identify novel and highly conserved regulators of sleep.

genetics↗

bioRxiv2025

Cross-species variant-to-function analyses implicate MEIS1 in conferring sleep abnormalities and impaired cerebellar development

Genome wide association study (GWAS) reports substantially outpace subsequent functional characterization. Pinpointing the causal effector gene(s) at GWAS loci remains challenging given the non-coding genomic residency of >98% of these signals. We previously implicated effector genes at GWAS loci for the complex and polygenic disorder of human insomnia using a high-resolution cell-specific, chromatin capture-based variant-to-gene mapping protocol, paired with sleep phenotyping in Drosophila. In this study, we leveraged a diurnal vertebrate model with higher genomic conservation, namely zebrafish, to screen our six highest confidence candidate genes and identify those whose loss-of-function impaired sleep characteristics related to human insomnia-like behaviors. Of these genes, we observed that CRISPR-mediated deletion of the zebrafish ortholog of MEIS1 produced nighttime specific sleep fragmentation and increased latency to sleep, pointing to a conserved role for MEIS1 in sleep maintenance. Comparing our human cell-based chromatin accessibility and contact maps with publicly available zebrafish spatial genomic data revealed highly conserved genomic architecture harboring the insomnia GWAS variant of interest. Notably, this genomic conservation was selective for the zebrafish ortholog which contributed to the sleep phenotype, meis1b, while the duplicated ohnolog meis1a proved dispensable. Motivated by this, we characterized the spatio-temporal expression of meis1b in zebrafish, showing it is comparable to human with respect to cerebellar granule progenitors. Ultimately, we found that loss of meis1b impairs cerebellar development. Together, our work provides a powerful model for screening human disorder risk genes for sleep fragmentation using a tractable vertebrate and supports a conserved cerebellar role for MEIS1 in sleep disturbance.

neuroscience↗

bioRxiv2024

Cross-species evidence for a developmental origin of adult hypersomnia with loss of synaptic adhesion molecules beat-Ia/CADM2

Idiopathic hypersomnia (IH) is a poorly-understood sleep disorder characterized by excessive daytime sleepiness despite normal nighttime sleep. Combining human genomics with behavioral and mechanistic studies in fish and flies, we uncover a role for beat-Ia/CADM2, synaptic adhesion molecules of the immunoglobulin superfamily, in excessive sleepiness. Neuronal knockdown of Drosophila beat-Ia results in sleepy flies and loss of the vertebrate ortholog of beat-Ia, CADM2, results in sleepy fish. We delineate a developmental function for beat-Ia in synaptic elaboration of neuropeptide F (NPF) neurites projecting to the suboesophageal zone (SEZ) of the fly brain. Brain connectome and experimental evidence demonstrate these NPF outputs synapse onto a subpopulation of SEZ GABAergic neurons to stabilize arousal. NPF is the Drosophila homolog of vertebrate neuropeptide Y (NPY), and an NPY receptor agonist restores sleep to normal levels in zebrafish lacking CADM2. These findings point towards NPY modulation as a treatment target for human hypersomnia.

neuroscience↗

bioRxiv2023

Perturbation of the insomnia WDR90 GWAS locus pinpoints rs3752495 as a causal variant influencing distal expression of neighboring gene, PIG-Q

Although genome wide association studies (GWAS) have been crucial for the identification of loci associated with sleep traits and disorders, the method itself does not directly uncover the underlying causal variants and corresponding effector genes. The overwhelming majority of such variants reside in non-coding regions and are therefore presumed to impact the activity of cis-regulatory elements, such as enhancers. Our previously reported variant-to-gene mapping effort in human induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs), combined with validation in both Drosophila and zebrafish, implicated PIG-Q as a functionally relevant gene at the insomnia WDR90 locus. However, importantly that effort did not characterize the corresponding underlying causal variant at this GWAS signal. Specifically, our genome-wide ATAC-seq and high-resolution promoter-focused Capture C datasets generated in this cell setting brought our attention to a shortlist of three tightly neighboring single nucleotide polymorphisms (SNPs) in strong linkage disequilibrium in a candidate intronic enhancer region of WDR90 that contacted the open PIG-Q promoter. The objective of this study was to investigate the influence of the proxy SNPs collectively and then individually on PIG-Q modulation and to pinpoint the causal "regulatory" variant among the three SNPs. Starting at a gross level perturbation, deletion of the entire region harboring all three SNPs in human iPSC-derived neural progenitor cells via CRISPR-Cas9 editing and subsequent RNA sequencing revealed expression changes in specific PIG-Q transcripts. Results from more refined individual luciferase reporter assays for each of the three SNPs in iPSCs revealed that the intronic region with the rs3752495 risk allele induced a [~]2.5-fold increase in luciferase expression (n=10). Importantly, rs3752495 also exhibited an allele specific effect, with the risk allele increasing the luciferase expression by [~]2-fold compared to the non-risk allele. In conclusion, our variant-to-function approach and subsequent in vitro validation implicates rs3752495 as a causal insomnia risk variant embedded at the WDR90-PIG-Q locus.

genomics↗

bioRxiv2023

Screening effects of HCN channel blockers on sleep/wake behavior in zebrafish

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels generate electrical rhythmicity in various tissues although primarily heart, retina and brain. The HCN channel blocker compound, Ivabradine (Corlanor), is approved by the US Food and Drug Administration (FDA) as a medication to lower heart rate by blocking hyperpolarization activated inward current in the sinoatrial node. In addition, a growing body of evidence suggests a role for HCN channels in regulation of sleep/wake behavior. Zebrafish larvae are ideal model organisms for high throughput drug screening, drug repurposing and behavioral phenotyping studies. We leveraged this model system to investigate effects of three HCN channel blockers (Ivabradine, Zatebradine Hydrochloride and ZD7288) at multiple doses on sleep/wake behavior in wild type zebrafish. Results of interest included shorter latency to day time sleep at 0.1 M dose of Ivabradine (ANOVA, p:0.02), moderate reductions in average activity at 30 M dose of Zatebradine Hydrochloride (ANOVA, p:0.024) in daytime, and increased nighttime sleep at 4.5 M dose of ZD7288 (ANOVA, p:0.036). These differences support the hypothesis that compounds blocking HCN channels decreases wakefulness. HighlightsO_LIA drug screening study in which effects of HCN channel blocker compounds were tested displayed decreased wakefulwness in zebrafish. C_LIO_LIThere was modest evidence of these drugs on sleep and wake phenotypes including shorter latency to sleep, moderate reductions in average activity and increased sleep at different doses of three compounds compared to DMSO. C_LIO_LIWhile several specific doses of Ivabradine, Zatebradine hydrochloride or ZD7288 demonstrated some differences compared to DMSO, effects of these compounds was smaller than the effect of melatonin, a positive control. C_LI

pharmacology and toxicology↗

bioRxiv2022

Zebrafish screen of high-confidence targets at insomnia GWAS loci implicates MEIS1, SKIV2L, and ARFGAP2 as conserved regulators of sleep-wake behaviors

Recent large-scale human genome-wide association studies (GWAS) for insomnia have identified more than 200 significant loci. The functional relevance of these loci to the pathogenesis of insomnia is largely unknown. GWAS signals are typically non-coding variants, which are often arbitrarily annotated to the nearest protein-coding gene; however, due to 3D chromatin structure, variants can interact with more distal genes driving their function. The distal gene may, therefore, represent the true causal gene influencing the phenotype. By integrating our high-resolution chromatin interaction maps from neural progenitor cells with phenotypic data from a Drosophila RNAi screen, we prioritized candidate genes that we hypothesized would have deep phylogenetic conservation of sleep function. To determine the conservation of these candidate genes in the context of vertebrate sleep and their relevance to insomnia-like behaviors, we performed CRISPR-Cas9 mutagenesis in larval zebrafish for six highly conserved candidate genes and examined sleep-wake behaviors using automated video-tracking. CRISPR mutation of zebrafish orthologs of MEIS1 and SKIV2L produced insomnia-like behaviors, while mutation of ARFGAP2 impaired activity and development in our larval zebrafish model, demonstrating the importance of performing functional validation of GWAS-implicated effector genes to reveal genes influencing disease-relevant mechanisms.

genetics↗

bioRxiv2021

Variant-to-gene-mapping followed by cross-species genetic screening identifies GPI-anchor biosynthesis as novel regulator of sleep

Sleep is nearly ubiquitous throughout the animal kingdom, with deficiencies in sleep having been linked to a wide range of human disorders and diseases. While genome wide association studies (GWAS) in humans have identified loci robustly associated with several heritable diseases or traits, little is known about the functional roles of the underlying causal variants in regulating sleep duration or quality. We applied an ATAC-seq/promoter focused Capture C strategy in human iPSC-derived neural progenitors to carry out a variant-to-gene mapping campaign that identified 88 candidate sleep effector genes connected to relevant GWAS signals. To functionally validate the role of the implicated effector genes in sleep regulation, we performed a neuron-specific RNAi screen in the fruit fly, Drosophila melanogaster. This approach identified a number of genes that regulated sleep, including phosphatidylinositol N-acetylglucosaminyltransferase subunit Q (PIG-Q), a gene that encodes an enzyme involved in the first step of glycosylphosphatidylinositol (GPI)- anchor biosynthesis. We show that flies deficient for PIG-Q have longer sleep during both day and night due to an increase in the total number of sleep bouts. Subsequent systematic investigation of other PIG-family genes identified increased sleep in flies for multiple different genes within the PIG pathway. We then mutated the PIG-Q locus in zebrafish and identified similar increases in sleep to those observed in Drosophila, confirming deep homology of PIG-Q mediated sleep regulation. These results provide the first physical variant-to-gene mapping of human sleep genes followed by a model organism-based prioritization, revealing a novel and conserved role for GPI-anchor biosynthesis in sleep regulation.

genetics↗

bioRxiv2019

Characterization of Genetic and Phenotypic Heterogeneity of Obstructive Sleep Apnea Using Electronic Health Records

Obstructive sleep apnea (OSA) is defined by frequent episodes of reduced or complete cessation of airflow during sleep and is linked to negative health outcomes. Understanding the genetic factors influencing expression of OSA may lead to new treatment strategies. Electronic health records can be leveraged to both validate previously reported OSA-associated genomic variation and detect novel relationships between these variants and comorbidities. We identified candidate single nucleotide polymorphisms (SNPs) via systematic literature review of existing research. Using datasets available at Geisinger (n=39,407) and Vanderbilt University Medical Center (n=24,084), we evaluated associations between 48 SNPs and OSA diagnosis, defined using clinical codes. We also evaluated associations between these SNPs and OSA severity measures obtained from sleep reports at Geisinger (n=6,571). Finally, we used a phenome-wide approach to perform discovery and replication analyses testing associations between OSA candidate SNPs and other clinical codes and laboratory values. Ten SNPs were associated with OSA diagnosis in at least one dataset, and one additional SNP was associated following meta-analysis across all datasets. Three other SNPs were solely associated in subgroups defined by established risk factors (i.e., age, sex, and BMI). Five OSA diagnosis-associated SNPs, and 16 additional SNPs, were associated with OSA severity measures. SNPs associated with OSA diagnosis were also associated with codes reflecting cardiovascular disease, diabetes, celiac disease, peripheral nerve disorders and genitourinary symptoms. Results highlight robust OSA-associated SNPs, and provide evidence of convergent mechanisms influencing risk for co-occurring conditions. This knowledge can lead to more personalized treatments for OSA and related comorbidities.

genetics↗

bioRxiv2018

Calculating the Effects of Autism Risk Gene Variants on Dysfunction of Biological Processes Identifies Clinically-Useful Information

Autism spectrum disorders (ASD) are neurodevelopmental conditions that are influenced by genetic factors and encompass a wide-range and severity of symptoms. The details of how genetic variation contributes to variable symptomatology are unclear, creating a major challenge for translating vast amounts of data into clinically-useful information. To determine if variation in ASD risk genes correlates with symptomatology differences among individuals with ASD, thus informing treatment, we developed an approach to calculate the likelihood of genetic dysfunction in Gene Ontology-defined biological processes that have significant overrepresentation of known risk genes. Using whole-exome sequence data from 2,381 individuals with ASD included in the Simons Simplex Collection, we identified likely damaging variants and conducted a clustering analysis to define subgroups based on scores reflecting genetic dysfunction in each process of interest to ASD etiology. Dysfunction in cognition-related genes distinguished a distinct subset of individuals with increased social deficits, lower IQs, and reduced adaptive behaviors when compared to individuals with no evidence of cognition-related gene dysfunction. In particular, a stop-gain variant in the pharmacogene encoding cycloxygenase-2 was associated with having an IQ<70 (i.e. intellectual disability), a key comorbidity in ASD. We expect that screening genes involved in cognition for deleterious variants in ASD cases may be useful for identifying clinically-informative factors that should be prioritized for functional follow-up. This has implications in designing more comprehensive genetic testing panels and may help provide the basis for more informed treatment in ASD.

genetics↗

bioRxiv2018

Estimating sleep parameters using an accelerometer without sleep diary

Wrist worn raw-data accelerometers are used increasingly in large scale population research. We examined whether sleep parameters can be estimated from these data in the absence of sleep diaries. Our heuristic algorithm uses the variance in estimated z-axis angle and makes basic assumptions about sleep interruptions. Detected sleep period time window (SPT-window), was compared against sleep diary in 3752 participants (range=60-82years) and polysomnography in sleep clinic patients (N=28) and in healthy good sleepers (N=22). The SPT-window derived from the algorithm was 10.9 and 2.9 minutes longer compared with sleep diary in men and women, respectively. Mean C-statistic to detect the SPT-window compared to polysomnography was 0.86 and 0.83 in clinic-based and healthy sleepers, respectively. We demonstrated the accuracy of our algorithm to detect the SPT-window. The value of this algorithm lies in studies such as UK Biobank where a sleep diary was not used.

epidemiology↗