Genome-wide association studies (GWAS) in humans have identified loci robustly associated with several heritable diseases or traits, yet 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 RNA interference screen in the fruit fly, Drosophila melanogaster , followed by validation in zebrafish. This approach identified a number of genes that regulate sleep including a critical role for glycosylphosphatidylinositol (GPI)–anchor biosynthesis. These results provide the first physical variant-to-gene mapping of human sleep genes followed by a model organism–based prioritization, revealing a conserved role for GPI-anchor biosynthesis in sleep regulation.

Essential tremor (ET) is one of the most common movement disorders. The etiology of ET remains largely unexplained. Whole genome sequencing (WGS) is likely to be of value in understanding a large proportion of ET with Mendelian and complex disease inheritance patterns. In ET families with Mendelian inheritance patterns, WGS may lead to gene identification where WES analysis failed to identify the causative single nucleotide variant (SNV) or indel due to incomplete coverage of the entire coding region of the genome, in addition to accurate detection of larger structural variants (SVs) and copy number variants (CNVs). Alternatively, in ET families with complex disease inheritance patterns with gene x gene and gene x environment interactions enrichment of functional rare coding and non-coding variants may explain the heritability of ET. We performed WGS in eight ET families (n = 40 individuals) enrolled in the Family Study of Essential Tremor. The analysis included filtering WGS data based on allele frequency in population databases, rare SNV and indel classification and association testing using the Mixed-Model Kernel Based Adaptive Cluster (MM-KBAC) test. A separate analysis of rare SV and CNVs segregating within ET families was also performed. Prioritization of candidate genes identified within families was performed using phenolyzer. WGS analysis identified candidate genes for ET in 5/8 (62.5%) of the families analyzed. WES analysis in a subset of these families in our previously published study failed to identify candidate genes. In one family, we identified a deleterious and damaging variant (c.1367G>A, p.(Arg456Gln)) in the candidate gene, CACNA1G, which encodes the pore forming subunit of T-type Ca(2+) channels, Ca_V3.1, and is expressed in various motor pathways and has been previously implicated in neuronal autorhythmicity and ET. Other candidate genes identified include SLIT3 which encodes an axon guidance molecule and in three families, phenolyzer prioritized genes that are associated with hereditary neuropathies (family A, KARS, family B, KIF5A and family F, NTRK1). Functional studies of CACNA1G and SLIT3 suggest a role for these genes in ET disease pathogenesis.

The C-promoter binding factor-1 (CBF-1) is a potent and specific inhibitor of the human immunodeficiency virus (HIV)-1 LTR promoter. Here, we demonstrate that the knockdown of endogenous CBF-1 in latently infected primary CD4+ T cells, using specific small hairpin RNAs (shRNA), resulted in the reactivation of latent HIV proviruses. Chromatin immunoprecipitation (ChIP) assays using latently infected primary T cells and Jurkat T-cell lines demonstrated that CBF-1 induces the establishment and maintenance of HIV latency by recruiting polycomb group (PcG/PRC) corepressor complexes or polycomb repressive complexes 1 and 2 (PRC1 and PRC2). Knockdown of CBF-1 resulted in the dissociation of PRCs corepressor complexes enhancing the recruitment of RNA polymerase II (RNAP II) at HIV LTR. Knockdown of certain components of PRC1 and PRC2 also led to the reactivation of latent proviruses. Similarly, the treatment of latently infected primary CD4+ T cells with the PRC2/EZH2 inhibitor, 3-deazaneplanocin A (DZNep), led to their reactivation.

Essential Tremor (ET) is one of the most common neurological diseases, with an estimated 7 million affected individuals in the US; the pathophysiology of the disorder is poorly understood. Recently, we identified a mutation (KCNS2 (Kv9.2), c.1137 T > A, p.(D379E) in an electrically silent voltage-gated K⁺ channel α-subunit, Kv9.2, in a family with ET, that modulates the activity of Kv2 channels. We have produced transgenic Drosophila lines that express either the human wild type Kv9.2 (hKv9.2) or the ET causing mutant Kv9.2 (hKv9.2-D379E) subunit in all neurons. We show that the hKv9.2 subunit modulates activity of endogenous Drosophila K⁺ channel Shab. The mutant hKv9.2-D379E subunit showed significantly higher levels of Shab inactivation and a higher frequency of spontaneous firing rate consistent with neuronal hyperexcitibility. We also observed behavioral manifestations of nervous system dysfunction including effects on night time activity and sleep. This functional data further supports the pathogenicity of the KCNS2 (p.D379E) mutation, consistent with our prior observations including co-segregation with ET in a family, a likely pathogenic change in the channel pore domain and absence from population databases. The Drosophila hKv9.2 transgenic model recapitulates several features of ET and may be employed to advance our understanding of ET disease pathogenesis.