The Genetics of Human Sleep and Sleep Disorders.

Healthy sleep is vital for humans to achieve optimal health and longevity. Poor sleep and sleep disorders are strongly associated with increased morbidity and mortality. However, the importance of good sleep continues to be underrecognized. Mechanisms regulating sleep and its functions in humans remain mostly unclear even after decades of dedicated research. Advancements in gene sequencing techniques and computational methodologies have paved the way for various genetic analysis approaches, which have provided some insights into human sleep genetics. This review summarizes our current knowledge of the genetic basis underlying human sleep traits and sleep disorders. We also highlight the use of animal models to validate genetic findings from human sleep studies and discuss potential molecular mechanisms and signaling pathways involved in the regulation of human sleep.

Diverse roles of pontine NPS-expressing neurons in sleep regulation.

Neuropeptide S (NPS) was postulated to be a wake-promoting neuropeptide with unknown mechanism, and a mutation in its receptor (NPSR1) causes the short sleep duration trait in humans. We investigated the role of different NPS+ nuclei in sleep/wake regulation. Loss-of-function and chemogenetic studies revealed that NPS+ neurons in the parabrachial nucleus (PB) are wake-promoting, whereas peri-locus coeruleus (peri-LC) NPS+ neurons are not important for sleep/wake modulation. Further, we found that a NPS+ nucleus in the central gray of the pons (CGPn) strongly promotes sleep. Fiber photometry recordings showed that NPS+ neurons are wake-active in the CGPn and wake/REM-sleep active in the PB and peri-LC. Blocking NPS-NPSR1 signaling or knockdown of Nps supported the function of the NPS-NPSR1 pathway in sleep/wake regulation. Together, these results reveal that NPS and NPS+ neurons play dichotomous roles in sleep/wake regulation at both the molecular and circuit levels.

Mutant ß1-adrenergic receptor improves REM sleep and ameliorates tau accumulation in a mouse model of tauopathy.

Sleep is essential for our well-being, and chronic sleep deprivation has unfavorable health consequences. We recently demonstrated that two familial natural short sleep (FNSS) mutations, DEC2-P384R and Npsr1-Y206H, are strong genetic modifiers of tauopathy in PS19 mice, a model of tauopathy. To gain more insight into how FNSS variants modify the tau phenotype, we tested the effect of another FNSS gene variant, Adrb1-A187V, by crossing mice with this mutation onto the PS19 background. We found that the Adrb1-A187V mutation helped restore rapid eye movement (REM) sleep and alleviated tau aggregation in a sleep-wake center, the locus coeruleus (LC), in PS19 mice. We found that ADRB1+ neurons in the central amygdala (CeA) sent projections to the LC, and stimulating CeAADRB1+ neuron activity increased REM sleep. Furthermore, the mutant Adrb1 attenuated tau spreading from the CeA to the LC. Our findings suggest that the Adrb1-A187V mutation protects against tauopathy by both mitigating tau accumulation and attenuating tau spreading.

Mutations in potassium channel Kir2.6 cause susceptibility to thyrotoxic hypokalemic periodic paralysis.

Thyrotoxic hypokalemic periodic paralysis (TPP) is characterized by acute attacks of weakness, hypokalemia, and thyrotoxicosis of various etiologies. These transient attacks resemble those of patients with familial hypokalemic periodic paralysis (hypoKPP) and resolve with treatment of the underlying hyperthyroidism. Because of the phenotypic similarity of these conditions, we hypothesized that TPP might also be a channelopathy. While sequencing candidate genes, we identified a previously unreported gene (not present in human sequence databases) that encodes an inwardly rectifying potassium (Kir) channel, Kir2.6. This channel, nearly identical to Kir2.2, is expressed in skeletal muscle and is transcriptionally regulated by thyroid hormone. Expression of Kir2.6 in mammalian cells revealed normal Kir currents in whole-cell and single-channel recordings. Kir2.6 mutations were present in up to 33% of the unrelated TPP patients in our collection. Some of these mutations clearly alter a variety of Kir2.6 properties, all altering muscle membrane excitability leading to paralysis.

An excitatory peri-tegmental reticular nucleus circuit for wake maintenance.

Sleep is a necessity for our survival, but its regulation remains incompletely understood. Here, we used a human sleep duration gene to identify a population of cells in the peri-tegmental reticular nucleus (pTRNADRB1) that regulate sleep-wake, uncovering a role for a poorly understood brain area. Although initial ablation in mice led to increased wakefulness, further validation revealed that pTRNADRB1 neuron stimulation strongly promotes wakefulness, even after stimulation offset. Using combinatorial genetics, we found that excitatory pTRNADRB1 neurons promote wakefulness. pTRN neurons can be characterized as anterior- or posterior-projecting neurons based on multiplexed analysis of projections by sequencing (MAPseq) analysis. Finally, we found that pTRNADRB1 neurons promote wakefulness, in part, through projections to the lateral hypothalamus. Thus, human genetic information from a human sleep trait allowed us to identify a role for the pTRN in sleep-wake regulation.

Adaptive Long-Read Sequencing Reveals GGC Repeat Expansion in ZFHX3 Associated with Spinocerebellar Ataxia Type 4.

BACKGROUND: Spinocerebellar ataxia type 4 (SCA4) is an autosomal dominant ataxia with invariable sensory neuropathy originally described in a family with Swedish ancestry residing in Utah more than 25 years ago. Despite tight linkage to the 16q22 region, the molecular diagnosis has since remained elusive. OBJECTIVES: Inspired by pathogenic structural variation implicated in other 16q-ataxias with linkage to the same locus, we revisited the index SCA4 cases from the Utah family using novel technologies to investigate structural variation within the candidate region. METHODS: We adopted a targeted long-read sequencing approach with adaptive sampling on the Oxford Nanopore Technologies (ONT) platform that enables the detection of segregating structural variants within a genomic region without a priori assumptions about any variant features. RESULTS: Using this approach, we found a heterozygous (GGC)n repeat expansion in the last coding exon of the zinc finger homeobox 3 (ZFHX3) gene that segregates with disease, ranging between 48 and 57 GGC repeats in affected probands. This finding was replicated in a separate family with SCA4. Furthermore, the estimation of this GGC repeat size in short-read whole genome sequencing (WGS) data of 21,836 individuals recruited to the 100,000 Genomes Project in the UK and our in-house dataset of 11,258 exomes did not reveal any pathogenic repeats, indicating that the variant is ultrarare. CONCLUSIONS: These findings support the utility of adaptive long-read sequencing as a powerful tool to decipher causative structural variation in unsolved cases of inherited neurological disease. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Striatal Indirect Pathway Dysfunction Underlies Motor Deficits in a Mouse Model of Paroxysmal Dyskinesia.

Abnormal involuntary movements, or dyskinesias, are seen in many neurologic diseases, including disorders where the brain appears grossly normal. This observation suggests that alterations in neural activity or connectivity may underlie dyskinesias. One influential model proposes that involuntary movements are driven by an imbalance in the activity of striatal direct and indirect pathway neurons (dMSNs and iMSNs, respectively). Indeed, in some animal models, there is evidence that dMSN hyperactivity contributes to dyskinesia. Given the many diseases associated with dyskinesia, it is unclear whether these findings generalize to all forms. Here, we used male and female mice in a mouse model of paroxysmal nonkinesigenic dyskinesia (PNKD) to assess whether involuntary movements are related to aberrant activity in the striatal direct and indirect pathways. In this model, as in the human disorder PNKD, animals experience dyskinetic attacks in response to caffeine or alcohol. Using optically identified striatal single-unit recordings in freely moving PNKD mice, we found a loss of iMSN firing during dyskinesia bouts. Further, chemogenetic inhibition of iMSNs triggered dyskinetic episodes in PNKD mice. Finally, we found that these decreases in iMSN firing are likely because of aberrant endocannabinoid-mediated suppression of glutamatergic inputs. These data show that striatal iMSN dysfunction contributes to the etiology of dyskinesia in PNKD, and suggest that indirect pathway hypoactivity may be a key mechanism for the generation of involuntary movements in other disorders.SIGNIFICANCE STATEMENT Involuntary movements, or dyskinesias, are part of many inherited and acquired neurologic syndromes. There are few effective treatments, most of which have significant side effects. Better understanding of which cells and patterns of activity cause dyskinetic movements might inform the development of new neuromodulatory treatments. In this study, we used a mouse model of an inherited human form of paroxysmal dyskinesia in combination with cell type-specific tools to monitor and manipulate striatal activity. We were able to narrow in on a specific group of neurons that causes dyskinesia in this model, and found alterations in a well-known form of plasticity in this cell type, endocannabinoid-dependent synaptic LTD. These findings point to new areas for therapeutic development.

TIMELESS mutation alters phase responsiveness and causes advanced sleep phase.

Many components of the circadian molecular clock are conserved from flies to mammals; however, the role of mammalian Timeless remains ambiguous. Here, we report a mutation in the human TIMELESS (hTIM) gene that causes familial advanced sleep phase (FASP). Tim CRISPR mutant mice exhibit FASP with altered photic entrainment but normal circadian period. We demonstrate that the mutation prevents TIM accumulation in the nucleus and has altered affinity for CRY2, leading to destabilization of PER/CRY complex and a shortened period in nonmature mouse embryonic fibroblasts (MEFs). We conclude that TIM, when excluded from the nucleus, can destabilize the negative regulators of the circadian clock, alter light entrainment, and cause FASP.

Episodic neurologic disorders: syndromes, genes, and mechanisms.

Many neurologic diseases cause discrete episodic impairment in contrast with progressive deterioration. The symptoms of these episodic disorders exhibit striking variety. Herein we review what is known of the phenotypes, genetics, and pathophysiology of episodic neurologic disorders. Of these, most are genetically complex, with unknown or polygenic inheritance. In contrast, a fascinating panoply of episodic disorders exhibit Mendelian inheritance. We classify episodic Mendelian disorders according to the primary neuroanatomical location affected: skeletal muscle, cardiac muscle, neuromuscular junction, peripheral nerve, or central nervous system (CNS). Most known Mendelian mutations alter genes that encode membrane-bound ion channels. These mutations cause ion channel dysfunction, which ultimately leads to altered membrane excitability as manifested by episodic disease. Other Mendelian disease genes encode proteins essential for ion channel trafficking or stability. These observations have cemented the channelopathy paradigm, in which episodic disorders are conceptualized as disorders of ion channels. However, we expand on this paradigm to propose that dysfunction at the synaptic and neuronal circuit levels may underlie some episodic neurologic entities.

A Mitochondrial tRNA Mutation Causes Axonal CMT in a Large Venezuelan Family.

OBJECTIVE: The objective of this study was to identify the genetic cause for progressive peripheral nerve disease in a Venezuelan family. Despite the growing list of genes associated with Charcot-Marie-Tooth disease, many patients with axonal forms lack a genetic diagnosis. METHODS: A pedigree was constructed, based on family clinical data. Next-generation sequencing of mitochondrial DNA (mtDNA) was performed for 6 affected family members. Muscle biopsies from 4 family members were used for analysis of muscle histology and ultrastructure, mtDNA sequencing, and RNA quantification. Ultrastructural studies were performed on sensory nerve biopsies from 2 affected family members. RESULTS: Electrodiagnostic testing showed a motor and sensory axonal polyneuropathy. Pedigree analysis revealed inheritance only through the maternal line, consistent with mitochondrial transmission. Sequencing of mtDNA identified a mutation in the mitochondrial tRNAVal (mt-tRNAVal ) gene, m.1661A>G, present at nearly 100% heteroplasmy, which disrupts a Watson-Crick base pair in the T-stem-loop. Muscle biopsies showed chronic denervation/reinnervation changes, whereas biochemical analysis of electron transport chain (ETC) enzyme activities showed reduction in multiple ETC complexes. Northern blots from skeletal muscle total RNA showed severe reduction in abundance of mt-tRNAVal , and mildly increased mt-tRNAPhe , in subjects compared with unrelated age- and sex-matched controls. Nerve biopsies from 2 affected family members demonstrated ultrastructural mitochondrial abnormalities (hyperplasia, hypertrophy, and crystalline arrays) consistent with a mitochondrial neuropathy. CONCLUSION: We identify a previously unreported cause of Charcot-Marie-Tooth (CMT) disease, a mutation in the mt-tRNAVal , in a Venezuelan family. This work expands the list of CMT-associated genes from protein-coding genes to a mitochondrial tRNA gene. ANN NEUROL 2020;88:830-842.

DEC2 modulates orexin expression and regulates sleep.

Adequate sleep is essential for physical and mental health. We previously identified a missense mutation in the human DEC2 gene (BHLHE41) leading to the familial natural short sleep behavioral trait. DEC2 is a transcription factor regulating the circadian clock in mammals, although its role in sleep regulation has been unclear. Here we report that prepro-orexin, also known as hypocretin (Hcrt), gene expression is increased in the mouse model expressing the mutant hDEC2 transgene (hDEC2-P384R). Prepro-orexin encodes a precursor protein of a neuropeptide producing orexin A and B (hcrt1 and hcrt2), which is enriched in the hypothalamus and regulates maintenance of arousal. In cell culture, DEC2 suppressed prepro-orexin promoter-luc (ore-luc) expression through cis-acting E-box elements. The mutant DEC2 has less repressor activity than WT-DEC2, resulting in increased orexin expression. DEC2-binding affinity for the prepro-orexin gene promoter is decreased by the P384R mutation, likely due to weakened interaction with other transcription factors. In vivo, the decreased immobility time of the mutant transgenic mice is attenuated by an orexin receptor antagonist. Our results suggested that DEC2 regulates sleep/wake duration, at least in part, by modulating the neuropeptide hormone orexin.

A PERIOD3 variant causes a circadian phenotype and is associated with a seasonal mood trait.

In humans, the connection between sleep and mood has long been recognized, although direct molecular evidence is lacking. We identified two rare variants in the circadian clock gene PERIOD3 (PER3-P415A/H417R) in humans with familial advanced sleep phase accompanied by higher Beck Depression Inventory and seasonality scores. hPER3-P415A/H417R transgenic mice showed an altered circadian period under constant light and exhibited phase shifts of the sleep-wake cycle in a short light period (photoperiod) paradigm. Molecular characterization revealed that the rare variants destabilized PER3 and failed to stabilize PERIOD1/2 proteins, which play critical roles in circadian timing. Although hPER3-P415A/H417R-Tg mice showed a mild depression-like phenotype, Per3 knockout mice demonstrated consistent depression-like behavior, particularly when studied under a short photoperiod, supporting a possible role for PER3 in mood regulation. These findings suggest that PER3 may be a nexus for sleep and mood regulation while fine-tuning these processes to adapt to seasonal changes.

Episodic and electrical nervous system disorders caused by nonchannel genes.

As noted in the separate introduction to this special topic section, episodic and electrical disorders can appear quite different clinically and yet share many overlapping features, including attack precipitants, therapeutic responses, natural history, and the types of genes that cause many of the genetic forms (i.e., ion channel genes). Thus, as we mapped and attempted to clone genes causing other episodic disorders, ion channels were always outstanding candidates when they mapped to the critical region of linkage in such a family. However, some of these disorders do not result from mutations in channels. This realization has opened up large and exciting new areas for the pathogenesis of these disorders. In some cases, the mutations occur in genes of unknown function or without understanding of molecular pathogenesis. Recently, emerging insights into a fascinating group of episodic movement disorders, the paroxysmal dyskinesias, and study of the causative genes and proteins are leading to the emerging concept of episodic electric disorders resulting from synaptic dysfunction. Much work remains to be done, but the field is evolving rapidly. As it does, we have come to realize that the molecular pathogenesis of electrical and episodic disorders is more complex than a scenario in which such disorders are simply due to mutations in the primary determinants of membrane excitability (channels).

Protein mutated in paroxysmal dyskinesia interacts with the active zone protein RIM and suppresses synaptic vesicle exocytosis.

Paroxysmal nonkinesigenic dyskinesia (PNKD) is an autosomal dominant episodic movement disorder precipitated by coffee, alcohol, and stress. We previously identified the causative gene but the function of the encoded protein remains unknown. We also generated a PNKD mouse model that revealed dysregulated dopamine signaling in vivo. Here, we show that PNKD interacts with synaptic active zone proteins Rab3-interacting molecule (RIM)1 and RIM2, localizes to synapses, and modulates neurotransmitter release. Overexpressed PNKD protein suppresses release, and mutant PNKD protein is less effective than wild-type at inhibiting exocytosis. In PNKD KO mice, RIM1/2 protein levels are reduced and synaptic strength is impaired. Thus, PNKD is a novel synaptic protein with a regulatory role in neurotransmitter release.

Microfluidic droplet enrichment for targeted sequencing.

Targeted sequence enrichment enables better identification of genetic variation by providing increased sequencing coverage for genomic regions of interest. Here, we report the development of a new target enrichment technology that is highly differentiated from other approaches currently in use. Our method, MESA (Microfluidic droplet Enrichment for Sequence Analysis), isolates genomic DNA fragments in microfluidic droplets and performs TaqMan PCR reactions to identify droplets containing a desired target sequence. The TaqMan positive droplets are subsequently recovered via dielectrophoretic sorting, and the TaqMan amplicons are removed enzymatically prior to sequencing. We demonstrated the utility of this approach by generating an average 31.6-fold sequence enrichment across 250 kb of targeted genomic DNA from five unique genomic loci. Significantly, this enrichment enabled a more comprehensive identification of genetic polymorphisms within the targeted loci. MESA requires low amounts of input DNA, minimal prior locus sequence information and enriches the target region without PCR bias or artifacts. These features make it well suited for the study of genetic variation in a number of research and diagnostic applications.

Very large G protein-coupled receptor 1 regulates myelin-associated glycoprotein via Gαs/Gαq-mediated protein kinases A/C.

VLGR1 (very large G protein-coupled receptor 1), also known as MASS1 (monogenic audiogenic seizure susceptible 1), is an orphan G protein-coupled receptor that contains a large extracellular N terminus with 35 calcium-binding domains. A truncating mutation in the Mass1 gene causes autosomal recessive, sound-induced seizures in the Frings mouse. However, the function of MASS1 and the mechanism underlying Frings mouse epilepsy are not known. Here, we found that MASS1 protein is enriched in the myelinated regions of the superior and inferior colliculi, critical areas for the initiation and propagation of audiogenic seizures. Using a panel of myelin antibodies, we discovered that myelin-associated glycoprotein (MAG) expression is dramatically decreased in Frings mice. MASS1 inhibits the ubiquitylation of MAG, thus enhancing the stability of this protein, and the calcium-binding domains of MASS1 are essential for this regulation. Furthermore, MASS1 interacts with Gαs/Gαq and activates PKA and PKC in response to extracellular calcium. Suppression of signaling by MASS1 RNAi or a specific inhibitor abrogates MAG up-regulation. We postulate that MASS1 senses extracellular calcium and activates cytosolic PKA/PKC pathways to regulate myelination by means of MAG protein stability in myelin-forming cells of the auditory pathway. Further work is required to determine whether MAG dysregulation is a cause or consequence of audiogenic epilepsy and whether there are other pathways regulated by MASS1.

MicroRNA-23a promotes myelination in the central nervous system.

Demyelinating disorders including leukodystrophies are devastating conditions that are still in need of better understanding, and both oligodendrocyte differentiation and myelin synthesis pathways are potential avenues for developing treatment. Overexpression of lamin B1 leads to leukodystrophy characterized by demyelination of the central nervous system, and microRNA-23 (miR-23) was found to suppress lamin B1 and enhance oligodendrocyte differentiation in vitro. Here, we demonstrated that miR-23a-overexpressing mice have increased myelin thickness, providing in vivo evidence that miR-23a enhances both oligodendrocyte differentiation and myelin synthesis. Using this mouse model, we explored possible miR-23a targets and revealed that the phosphatase and tensin homologue/phosphatidylinositol trisphosphate kinase/Akt/mammalian target of rapamycin pathway is modulated by miR-23a. Additionally, a long noncoding RNA, 2700046G09Rik, was identified as a miR-23a target and modulates phosphatase and tensin homologue itself in a miR-23a-dependent manner. The data presented here imply a unique role for miR-23a in the coordination of proteins and noncoding RNAs in generating and maintaining healthy myelin.

Dual roles of FBXL3 in the mammalian circadian feedback loops are important for period determination and robustness of the clock.

The mammalian circadian clock is composed of interlocking feedback loops. Cryptochrome is a central component in the core negative feedback loop, whereas Rev-Erbα, a member of the nuclear receptor family, is an essential component of the interlocking loop. To understand the roles of different clock genes, we conducted a genetic interaction screen by generating single- and double-mutant mice. We found that the deletion of Rev-erbα in F-box/leucine rich-repeat protein (Fbxl3)-deficient mice rescued its long-circadian period phenotype, and our results further revealed that FBXL3 regulates Rev-Erb/retinoic acid receptor-related orphan receptor-binding element (RRE)-mediated transcription by inactivating the Rev-Erbα:histone deacetylase 3 corepressor complex. By analyzing the Fbxl3 and Cryptochrome 1 double-mutant mice, we found that FBXL3 also regulates the amplitudes of E-box-driven gene expression. These two separate roles of FBXL3 in circadian feedback loops provide a mechanism that contributes to the period determination and robustness of the clock.

Solving the mystery of human sleep schedules one mutation at a time.

Sleep behavior remains one of the most enigmatic areas of life. The unanswered questions range from "why do we sleep?" to "how we can improve sleep in today's society?" Identification of mutations responsible for altered circadian regulation of human sleep lead to unique opportunities for probing these territories. In this review, we summarize causative circadian mutations found from familial genetic studies to date. We also describe how these mutations mechanistically affect circadian function and lead to altered sleep behaviors, including shifted or shortening of sleep patterns. In addition, we discuss how the investigation of mutations can not only expand our understanding of the molecular mechanisms regulating the circadian clock and sleep duration, but also bridge the pathways between clock/sleep and other human physiological conditions and ailments such as metabolic regulation and migraine headaches.