Population-scale tissue transcriptomics maps long non-coding RNAs to complex disease.

Long non-coding RNA (lncRNA) genes have well-established and important impacts on molecular and cellular functions. However, among the thousands of lncRNA genes, it is still a major challenge to identify the subset with disease or trait relevance. To systematically characterize these lncRNA genes, we used Genotype Tissue Expression (GTEx) project v8 genetic and multi-tissue transcriptomic data to profile the expression, genetic regulation, cellular contexts, and trait associations of 14,100 lncRNA genes across 49 tissues for 101 distinct complex genetic traits. Using these approaches, we identified 1,432 lncRNA gene-trait associations, 800 of which were not explained by stronger effects of neighboring protein-coding genes. This included associations between lncRNA quantitative trait loci and inflammatory bowel disease, type 1 and type 2 diabetes, and coronary artery disease, as well as rare variant associations to body mass index.

Genomic Analysis in the Age of Human Genome Sequencing.

Affordable genome sequencing technologies promise to revolutionize the field of human genetics by enabling comprehensive studies that interrogate all classes of genome variation, genome-wide, across the entire allele frequency spectrum. Ongoing projects worldwide are sequencing many thousands-and soon millions-of human genomes as part of various gene mapping studies, biobanking efforts, and clinical programs. However, while genome sequencing data production has become routine, genome analysis and interpretation remain challenging endeavors with many limitations and caveats. Here, we review the current state of technologies for genetic variant discovery, genotyping, and functional interpretation and discuss the prospects for future advances. We focus on germline variants discovered by whole-genome sequencing, genome-wide functional genomic approaches for predicting and measuring variant functional effects, and implications for studies of common and rare human disease.

Ventral Pallidal GABAergic Neuron Calcium Activity Encodes Cue-Driven Reward Seeking and Persists in the Absence of Reward Delivery.

Reward-seeking behavior is often initiated by environmental cues that signal reward availability. This is a necessary behavioral response; however, cue reactivity and reward-seeking behavior can become maladaptive. To better understand how cue-elicited reward seeking becomes maladaptive, it is important to understand the neural circuits involved in assigning appetitive value to rewarding cues and actions. Ventral pallidum (VP) neurons are known to contribute to cue-elicited reward-seeking behavior and have heterogeneous responses in a discriminative stimulus (DS) task. The VP neuronal subtypes and output pathways that encode distinct aspects of the DS task remain unknown. Here, we used an intersectional viral approach with fiber photometry to record bulk calcium activity in VP GABAergic (VP GABA) neurons in male and female rats as they learned and performed the DS task. We found that VP GABA neurons are excited by reward-predictive cues but not neutral cues and that this response develops over time. We also found that this cue-evoked response predicts reward-seeking behavior and that inhibiting this VP GABA activity during cue presentation decreases reward-seeking behavior. Additionally, we found increased VP GABA calcium activity at the time of expected reward delivery, which occurred even on trials when reward was omitted. Together, these findings suggest that VP GABA neurons encode reward expectation, and calcium activity in these neurons encodes the vigor of cue-elicited reward seeking.SIGNIFICANCE STATEMENT VP circuitry is a major driver of cue-evoked behaviors. Previous work has found that VP neurons have heterogenous responses and contributions to reward-seeking behavior. This functional heterogeneity is because of differences of neurochemical subtypes and projections of VP neurons. Understanding the heterogenous responses among and within VP neuronal cell types is a necessary step in further understanding how cue-evoked behavior becomes maladaptive. Our work explores the canonical GABAergic VP neuron and how the calcium activity of these cells encodes components of cue-evoked reward seeking, including the vigor and persistence of reward seeking.

Association of structural variation with cardiometabolic traits in Finns.

The contribution of genome structural variation (SV) to quantitative traits associated with cardiometabolic diseases remains largely unknown. Here, we present the results of a study examining genetic association between SVs and cardiometabolic traits in the Finnish population. We used sensitive methods to identify and genotype 129,166 high-confidence SVs from deep whole-genome sequencing (WGS) data of 4,848 individuals. We tested the 64,572 common and low-frequency SVs for association with 116 quantitative traits and tested candidate associations using exome sequencing and array genotype data from an additional 15,205 individuals. We discovered 31 genome-wide significant associations at 15 loci, including 2 loci at which SVs have strong phenotypic effects: (1) a deletion of the ALB promoter that is greatly enriched in the Finnish population and causes decreased serum albumin level in carriers (p = 1.47 × 10-54) and is also associated with increased levels of total cholesterol (p = 1.22 × 10-28) and 14 additional cholesterol-related traits, and (2) a multi-allelic copy number variant (CNV) at PDPR that is strongly associated with pyruvate (p = 4.81 × 10-21) and alanine (p = 6.14 × 10-12) levels and resides within a structurally complex genomic region that has accumulated many rearrangements over evolutionary time. We also confirmed six previously reported associations, including five led by stronger signals in single nucleotide variants (SNVs) and one linking recurrent HP gene deletion and cholesterol levels (p = 6.24 × 10-10), which was also found to be strongly associated with increased glycoprotein level (p = 3.53 × 10-35). Our study confirms that integrating SVs in trait-mapping studies will expand our knowledge of genetic factors underlying disease risk.

Structural variants are a major source of gene expression differences in humans and often affect multiple nearby genes.

Structural variants (SVs) are an important source of human genome diversity, but their functional effects are poorly understood. We mapped 61,668 SVs in 613 individuals from the GTEx project and measured their effects on gene expression. We estimate that common SVs are causal at 2.66% of eQTLs, a 10.5-fold enrichment relative to their abundance in the genome. Duplications and deletions were the most impactful variant types, whereas the contribution of mobile element insertions was small (0.12% of eQTLs, 1.9-fold enriched). Multitissue analysis of eQTLs revealed that gene-altering SVs show more constitutive effects than other variant types, with 62.09% of coding SV-eQTLs active in all tissues with eQTL activity compared with 23.08% of coding SNV- and indel-eQTLs. Noncoding SVs, SNVs and indels show broadly similar patterns. We also identified 539 rare SVs associated with nearby gene expression outliers. Of these, 62.34% are noncoding SVs that affect gene expression but have modest enrichment at regulatory elements, showing that rare noncoding SVs are a major source of gene expression differences but remain difficult to predict from current annotations. Both common and rare SVs often affect the expression of multiple genes: SV-eQTLs affect an average of 1.82 nearby genes, whereas SNV- and indel-eQTLs affect an average of 1.09 genes, and 21.34% of rare expression-altering SVs show effects on two to nine different genes. We also observe significant effects on rare gene expression changes extending 1 Mb from the SV. This provides a mechanism by which individual SVs may have strong or pleiotropic effects on phenotypic variation.

The impact of rare variation on gene expression across tissues.

Rare genetic variants are abundant in humans and are expected to contribute to individual disease risk. While genetic association studies have successfully identified common genetic variants associated with susceptibility, these studies are not practical for identifying rare variants. Efforts to distinguish pathogenic variants from benign rare variants have leveraged the genetic code to identify deleterious protein-coding alleles, but no analogous code exists for non-coding variants. Therefore, ascertaining which rare variants have phenotypic effects remains a major challenge. Rare non-coding variants have been associated with extreme gene expression in studies using single tissues, but their effects across tissues are unknown. Here we identify gene expression outliers, or individuals showing extreme expression levels for a particular gene, across 44 human tissues by using combined analyses of whole genomes and multi-tissue RNA-sequencing data from the Genotype-Tissue Expression (GTEx) project v6p release. We find that 58% of underexpression and 28% of overexpression outliers have nearby conserved rare variants compared to 8% of non-outliers. Additionally, we developed RIVER (RNA-informed variant effect on regulation), a Bayesian statistical model that incorporates expression data to predict a regulatory effect for rare variants with higher accuracy than models using genomic annotations alone. Overall, we demonstrate that rare variants contribute to large gene expression changes across tissues and provide an integrative method for interpretation of rare variants in individual genomes.

Mitochondrial genome copy number measured by DNA sequencing in human blood is strongly associated with metabolic traits via cell-type composition differences.

BACKGROUND: Mitochondrial genome copy number (MT-CN) varies among humans and across tissues and is highly heritable, but its causes and consequences are not well understood. When measured by bulk DNA sequencing in blood, MT-CN may reflect a combination of the number of mitochondria per cell and cell-type composition. Here, we studied MT-CN variation in blood-derived DNA from 19184 Finnish individuals using a combination of genome (N = 4163) and exome sequencing (N = 19034) data as well as imputed genotypes (N = 17718). RESULTS: We identified two loci significantly associated with MT-CN variation: a common variant at the MYB-HBS1L locus (P = 1.6 × 10-8), which has previously been associated with numerous hematological parameters; and a burden of rare variants in the TMBIM1 gene (P = 3.0 × 10-8), which has been reported to protect against non-alcoholic fatty liver disease. We also found that MT-CN is strongly associated with insulin levels (P = 2.0 × 10-21) and other metabolic syndrome (metS)-related traits. Using a Mendelian randomization framework, we show evidence that MT-CN measured in blood is causally related to insulin levels. We then applied an MT-CN polygenic risk score (PRS) derived from Finnish data to the UK Biobank, where the association between the PRS and metS traits was replicated. Adjusting for cell counts largely eliminated these signals, suggesting that MT-CN affects metS via cell-type composition. CONCLUSION: These results suggest that measurements of MT-CN in blood-derived DNA partially reflect differences in cell-type composition and that these differences are causally linked to insulin and related traits.

When to test fetuses for RASopathies? Proposition from a systematic analysis of 352 multicenter cases and a postnatal cohort.

PURPOSE: Recent studies have identified suggestive prenatal features of RASopathies (e.g., increased nuchal translucency [NT], cystic hygroma [CH], hydrops, effusions, congenital heart diseases [CHD], polyhydramnios, renal anomalies). Our objective is to clarify indications for RASopathy prenatal testing. We compare genotype distributions between pre- and postnatal populations and propose genotype-phenotype correlations. METHODS: Three hundred fifty-two chromosomal microarray-negative cases sent for prenatal RASopathy testing between 2012 and 2019 were collected. For most, 11 RASopathy genes were tested. Postnatal cohorts (25 patients with available prenatal information and 108 institutional database genotypes) and the NSeuroNet database were used for genotypic comparisons. RESULTS: The overall diagnostic yield was 14% (50/352), with rates >20% for effusions, hydrops, and CHD. Diagnostic yield was significantly improved in presence of hypertrophic cardiomyopathy (HCM), persistent or associated CH, any suggestive finding combined with renal anomaly or polyhydramnios, or ≥2 ultrasound findings. Largest prenatal contributors of pathogenic variants were PTPN11 (30%), RIT1 (16%), RAF1 (14%), and HRAS (12%), which considerably differ from their prevalence in postnatal populations. HRAS, LZTR1, and RAF1 variants correlated with hydrops/effusions, and RIT1 with prenatal onset HCM. CONCLUSION: After normal chromosomal microarray, RASopathies should be considered when any ultrasound finding of lymphatic dysplasia or suggestive CHD is found alone or in association.

The migraine signature study: Methods and baseline results.

OBJECTIVE: To characterize patients who utilize services for migraine in a large integrated health care network, and describe patterns of care and utilization. BACKGROUND: Within health care systems, migraine is a common reason for seeking primary and neurology care, but relatively little is documented about who seeks care and the factors that explain variation in utilization. METHODS: We conducted a retrospective cohort study using electronic health record (EHR) data from Sutter Health primary care (PC) patients who had at least one office visit to a PC clinic between 2013 and 2017. Migraine status was ascertained from diagnosis codes and medication orders. Control status was assigned to those with no evidence of care for any type of headache. We divided the primary care migraine cohort into two groups: those who received all their care for migraine from PC (denoted PC-M) and those who had ≥1 encounter with a neurologist for migraine (denoted N-M). Migraine cases were also designated as having preexisting migraine if they had an encounter with a migraine diagnosis within (±) 6 months of their first study period PC visit and, otherwise, designated as first migraine consult. Two levels of contrasts included: patients with migraine and controls; and within the group of patients with migraine, PC-M and N-M groups. Comorbid conditions were determined from EHR encounter diagnosis codes. RESULTS: We identified 94,149 patients with migraine (including 21,525 N-M and 72,624 PC-M) and 1,248,763 controls. Comorbidities: Proportions of psychiatric [29.8% (n = 28,054) vs. 11.8% (n = 147,043)], autoimmune [(4.4% (n = 4162) vs. 2.6% (n = 31,981)], pain [13.2% (n = 12,439) vs. 5.8% (n = 72,049)], respiratory [24.6% (n = 23,186) vs. 12.3% (n = 153,692)], neurologic [2.9% (n = 2688) vs. 0.9% (n = 11,321)], and cerebrovascular [1.0% (n = 945) vs. 0.6% (n = 7500)] conditions were higher in the migraine group compared to controls, all p < 0.001. Among patients with migraine, the N-M group was similar to the PC-M group in sex, age, ethnicity, and marital status, but were more likely to have preexisting migraine (49.9% (n = 10,734) vs. 36.2% (n = 26,317), p < 0.001). Proportions of comorbid conditions were higher among the N-M group than the PC-M group {psychiatric [38.5% (n = 8291) vs. 27.2% (n = 19,763)], autoimmune [6.3% (n = 1365) vs. 3.9% (n = 2797)], pain [19.6% (n = 4218) vs. 11.3% (n = 8211)], respiratory [30.3% (n = 6516) vs. 23.0% (n = 16,670)], neurologic [6.0% (n = 1288) vs. 1.9% (n = 1400)], cardiovascular [9.7% (n = 2091) vs. 7.0% (n = 5076)], and cerebrovascular [2.3% (n = 500) vs. 0.6% (n = 445)], all p < 0.001}. Medications: During the study period, 82.6% (n = 77,762) of patients with migraine received ≥1 prescription order for an acute migraine medication [89.4% (n = 19,250) of N-M vs. 80.6% (n = 58,512) of PC]. Opioids were prescribed to 52.9% (n = 49,837) of patients with migraine [63.5% (n = 13,669) for N-M and 49.8% (n = 36,168) for PC-M patients). During the study period, 61.4% (n = 57,810) of patients received ≥1 prescription for a migraine preventive medication [81.4% (n = 17,521) of N-M and 55.5% (n = 40,289) of PC-M patients]. The most commonly prescribed classes of preventive medications were antidepressants. CONCLUSIONS: Among patients with migraine in a large health system, those who were also cared for in neurology were more likely to receive both acute and preventive medication migraine orders than those patients who did not see a neurologist, with triptans and antidepressants the most commonly prescribed classes of acute and preventive pharmacotherapies, respectively. Opioids were prescribed to approximately half of the total sample and more common in the N-M group. Adjusting for demographics, patients with migraine had higher rates of nearly every comorbidity we assessed and were more likely to utilize services compared to those without migraine. Overall, patients with migraine also cared for in neurology practices used more of all health care resource types under consideration and had more medical issues, which may be due in some part to a more severe, frequent and disabling disease state compared to those who sought care exclusively from PC practices.

Racial and Ethnic Disparities in Opioid Prescribing for Long Bone Fractures at Discharge From the Emergency Department: A Cross-sectional Analysis of 22 Centers From a Health Care Delivery System in Northern California.

STUDY OBJECTIVE: We examine racial and ethnic differences in opioid prescribing and dosing for long bone fractures at emergency department (ED) discharge. METHODS: We conducted an electronic health records-based cross-sectional study of adults with long bone fractures who presented to the ED across 22 sites from a health care delivery system (2016 to 2017). We examined differences in opioid prescribing at ED discharge and, among patients with a prescription, differences in opioid dosing (measured as morphine milligram equivalents) by race/ethnicity, using regression modeling with statistical adjustment for patient, fracture, and prescriber characteristics. RESULTS: A total of 11,576 patients with long bone fractures were included in the study; 64.4% were non-Hispanic white; 16.4%, 7.3%, 5.8%, and 5.1%, respectively, were Hispanic, Asian, black, and of other or unknown race; and 65.6% received an opioid at discharge. After adjusting for other factors, rates of opioid prescribing were not different by race/ethnicity; however, among patients with an opioid prescription, total morphine milligram equivalent units prescribed were 4.3%, 6.0%, and 8.1% less for Hispanics, blacks, and Asians relative to non-Hispanic whites. CONCLUSION: Racial and ethnic minority groups with long bone fractures receive similar frequencies of opioid prescriptions at discharge, with a small potency difference. How this affects pain relief and why it happens is unclear.

Proper formation of whisker barrelettes requires periphery-derived Smad4-dependent TGF-beta signaling.

Mammalian somatosensory topographic maps contain specialized neuronal structures that precisely recapitulate the spatial pattern of peripheral sensory organs. In the mouse, whiskers are orderly mapped onto several brainstem nuclei as a set of modular structures termed barrelettes. Using a dual-color iontophoretic labeling strategy, we found that the precise topography of barrelettes is not a result of ordered positions of sensory neurons within the ganglion. We next explored another possibility that formation of the whisker map is influenced by periphery-derived mechanisms. During the period of peripheral sensory innervation, several TGF-ß ligands are exclusively expressed in whisker follicles in a dynamic spatiotemporal pattern. Disrupting TGF-ß signaling, specifically in sensory neurons by conditional deletion of Smad4 at the late embryonic stage, results in the formation of abnormal barrelettes in the principalis and interpolaris brainstem nuclei and a complete absence of barrelettes in the caudalis nucleus. We further show that this phenotype is not derived from defective peripheral innervation or central axon outgrowth but is attributable to the misprojection and deficient segregation of trigeminal axonal collaterals into proper barrelettes. Furthermore, Smad4-deficient neurons develop simpler terminal arbors and form fewer synapses. Together, our findings substantiate the involvement of whisker-derived TGF-ß/Smad4 signaling in the formation of the whisker somatotopic maps.

Ventral pallidal GABAergic neurons drive consumption in male, but not female rats.

Food intake is controlled by multiple converging signals: hormonal signals that provide information about energy homeostasis, but also hedonic and motivational aspects of food and food cues that can drive non-homeostatic or "hedonic "feeding. The ventral pallidum (VP) is a brain region implicated in the hedonic and motivational impact of food and foods cues, as well as consumption of rewards. Disinhibition of VP neurons has been shown to generate intense hyperphagia, or overconsumption. While VP gamma-Aminobutyric acidergic (GABA) neurons have been implicated in cue-elicited reward seeking and motivation, the role of these neurons in the hyperphagia resulting from VP activation remains unclear. Here, we used Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to activate or inhibit VP GABA neurons in sated male and female rats during chow and sucrose consumption. We found that activation of VP GABA neurons increases consumption of chow and sucrose in male rats, but not female rats. We also found that, while inhibition of VP GABA neurons tended to decrease sucrose consumption, this effect was not statistically significant. Together, these findings suggest that activation of VP GABA neurons can stimulate consumption of routine or highly palatable rewards selectively in male rats.

Ventral pallidum neurons projecting to the ventral tegmental area reinforce but do not invigorate reward-seeking behavior.

Reward-predictive cues acquire motivating and reinforcing properties that contribute to the escalation and relapse of drug use in addiction. The ventral pallidum (VP) and ventral tegmental area (VTA) are two key nodes in brain reward circuitry implicated in addiction and cue-driven behavior. In the current study, we use in vivo fiber photometry and optogenetics to record from and manipulate VP→VTA in rats performing a discriminative stimulus task to determine the role these neurons play in invigoration and reinforcement by reward cues. We find that VP→VTA neurons are active during reward consumption and that optogenetic stimulation of these neurons biases choice behavior and is reinforcing. Critically, we find no encoding of reward-seeking vigor, and optogenetic stimulation does not enhance the probability or vigor of reward seeking in response to cues. Our results suggest that VP→VTA activity is more important for reinforcement than for invigoration of reward seeking by cues.

Phylogenomic analysis of bacterial and archaeal sequences with AMPHORA2.

SUMMARY: With the explosive growth of bacterial and archaeal sequence data, large-scale phylogenetic analyses present both opportunities and challenges. Here we describe AMPHORA2, an automated phylogenomic inference tool that can be used for high-throughput, high-quality genome tree reconstruction and metagenomic phylotyping. Compared with its predecessor, AMPHORA2 has several major enhancements and new functions: it has a greatly expanded phylogenetic marker database and can analyze both bacterial and archaeal sequences; it incorporates probability-based sequence alignment masks that improve the phylogenetic accuracy; it can analyze DNA as well as protein sequences and is more sensitive in marker identification; finally, it is over 100× faster in metagenomic phylotyping. AVAILABILITY: http://wolbachia.biology.virginia.edu/WuLab/Software.html. CONTACT: mw4yv@virginia.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Characterizing obesity in a large health care delivery system.

OBJECTIVES: To characterize the prevalence of obesity and associated health care use within an integrated health care system in California. STUDY DESIGN: Cross-sectional study using electronic health records. METHODS: Primary care patients 18 years and older receiving care at Sutter Health between 2015 and 2020 were included in the study. Obesity was classified and health care utilization was ascertained at index and during the follow-up periods. Differences in prevalence by demographic and clinical characteristics among patients with and without obesity were assessed. Logistic regression was used to estimate the relationship between obesity class and health care utilization (outpatient encounters). RESULTS: Of the 1,094,790 primary care patients included in the analysis, 35% were classified as having obesity, defined as a body mass index of 30 kg/m2 or more or 25 kg/m2 or more for Asian individuals. Obesity prevalence was greater in Hispanic patients (46%) than in non-Hispanic White patients (30%). Patients without obesity had fewer outpatient visits (mean [SD], 3.7 [3.8]) than those with class 1 (4.1 [4.0]), class 2 (4.6 [4.4]), and class 3 (5.2 [4.8]) obesity. In the fully adjusted regression model, the odds of being a high utilizer among patients with obesity were 1.1 (class 1), 1.2 (class 2), and 1.3 (class 3) times that of patients without obesity (P < .001). CONCLUSION: Obesity prevalence is high among patients in the Sutter Health system, varying by race/ethnicity, and was associated with increased outpatient visit utilization. There is a need for greater awareness of the impact of obesity and the specific patient populations affected by the disease.

Transcription factor short stature homeobox 2 is required for proper development of tropomyosin-related kinase B-expressing mechanosensory neurons.

Dorsal root ganglia (DRG) contain somatosensory neurons of diverse sensory modalities. Among these different types of sensory neurons, the molecular mechanisms that regulate the development and specification of touch neurons are the least well understood. We took a candidate approach and searched for transcription factors that are expressed in subsets of DRG neurons, and found that the transcription factor Shox2 (short stature homeobox 2) is expressed in subpopulations of TrkB (tropomyosin-related kinase B)- and Ret-expressing neurons at neonatal stages. Since TrkB is a known marker that is selectively expressed in touch sensory neurons, we decided to examine the function of Shox2 in specifying TrkB-positive DRG neurons. Conditional deletion of Shox2 in neural crest cells (which give rise to all DRG neurons) caused a 60 ∼ 65% reduction in the number of TrkB-expressing neurons. It also resulted in an increase in coexpression of TrkC in Ret-positive sensory neurons. Deletion of Shox2 in differentiating DRG neurons at later time points caused only a moderate reduction in TrkB expression. Overexpression of Shox2 in all neural crest cells resulted in a small increase in the number of TrkB-expressing neurons. Finally, Shox2 deletion also caused reduced touch sensory axonal innervation to layers III/IV of the spinal cord. Together, our findings identify Shox2 as an essential but not sufficient component of the transcription programs required in neural progenitor cells for the proper specification of subsets of TrkB-expressing touch/mechanosensory neurons.

Anxiety-like behavior and anxiolytic treatment in the Rett syndrome natural history study.

BACKGROUND: Rett syndrome (RTT) is a neurodevelopmental disorder most often related to a pathogenic variant in the X-linked MECP2 gene. Internalizing behaviors appear to be common, but standard methods of diagnosing anxiety are not readily applied in this population which typically has cognitive impairment and limited expressive language. This study aims to describe the frequency of anxiety-like behavior and anxiolytic treatments along with associated clinical features in individuals with RTT. METHODS: Parental reports and medication logs provided data from 1380 females with RTT participating in two iterations of the multicenter U.S. RTT Natural History Study (RNHS) from 2006 to 2019. RESULTS: Most participants with RTT (77.5%) had at least occasional anxious or nervous behavior. Anxiety was reported to be the most troublesome concern for 2.6%, and within the top 3 concerns for 10.0%, of participants in the second iteration. Parents directly reported treatment for anxious or nervous behavior in 16.6% of participants in the second iteration with most reporting good control of the behavior (71.6%). In the medication logs of both RNHS iterations, the indication of anxiety was listed for a similar number of participants (15% and 14.5%, respectively). Increased use of anxiolytics and selective serotonin reuptake inhibitors (SSRIs) was related to more frequent anxiety-like behaviors (P < 0.001), older age (P < 0.001), and mild MECP2 variants (P = 0.002). CONCLUSION: Anxiety-like behavior is frequent at all ages and is a significant parental concern in RTT. Older individuals and those with mild MECP2 variants are more likely to be treated with medications. Better diagnosis and treatment of anxiety in RTT should be a goal of both future studies and clinical care. TRIAL REGISTRATION: NCT00299312 and NCT02738281.

Transcriptomic signatures across human tissues identify functional rare genetic variation.

Rare genetic variants are abundant across the human genome, and identifying their function and phenotypic impact is a major challenge. Measuring aberrant gene expression has aided in identifying functional, large-effect rare variants (RVs). Here, we expanded detection of genetically driven transcriptome abnormalities by analyzing gene expression, allele-specific expression, and alternative splicing from multitissue RNA-sequencing data, and demonstrate that each signal informs unique classes of RVs. We developed Watershed, a probabilistic model that integrates multiple genomic and transcriptomic signals to predict variant function, validated these predictions in additional cohorts and through experimental assays, and used them to assess RVs in the UK Biobank, the Million Veterans Program, and the Jackson Heart Study. Our results link thousands of RVs to diverse molecular effects and provide evidence to associate RVs affecting the transcriptome with human traits.

ERαΔ4, an ERα splice variant missing exon4, interacts with caveolin-3 and mGluR2/3.

The two isoforms of the nuclear estrogen receptor, ERα and ERß are widely expressed in the central nervous system. Although they were first described as nuclear receptors, both isoforms have also been found at the cell membrane where they mediate cell signaling. Surface biotinylation studies using neuronal and glial primary cultures label an alternatively spliced form of ERα. The 52 kDa protein, ERαΔ4, is missing exon 4 and is highly expressed in membrane fractions derived from cultured cells. In vivo, both full-length (66 kDa) ERα and ERαΔ4 are present in membrane fractions. In response to estradiol, full-length ERα and ERαΔ4 are initially trafficked to the membrane, and then internalized in parallel. Previous studies determined that only the full-length ERα associates with metabotropic glutamate receptor-1a (mGluR1a), initiating cellular signaling. The role of ERαΔ4, remained to be elucidated. Here, we report ERαΔ4 trafficking, association with mGluR2/3, and downstream signaling in female rat arcuate nucleus (ARH). Caveolin (CAV) proteins are needed for ER transport to the cell membrane, and using co-immunoprecipitation CAV-3 was shown to associate with ERαΔ4. CAV-3 was necessary for ERαΔ4 trafficking to the membrane: in the ARH, microinjection of CAV-3 siRNA reduced CAV-3 and ERαΔ4a in membrane fractions by 50%, and 60%, respectively. Moreover, co-immunoprecipitation revealed that ERαΔ4 associated with inhibitory mGluRs, mGluR2/3. Estrogen benzoate (EB) treatment (5 µg; s.c.; every 4 days; three cycles) reduced levels of cAMP, an effect attenuated by antagonizing mGluR2/3. Following EB treatment, membrane levels of ERαΔ4 and mGluR2/3 were reduced implying ligand-induced internalization. These results implicate ERαΔ4 in an estradiol-induced inhibitory cell signaling in the ARH.

Behavioral profiles in Rett syndrome: Data from the natural history study.

INTRODUCTION: Rett syndrome (RTT) is a complex neurodevelopmental disorder with known behavioral abnormalities, both internalizing (e.g., anxiety, social withdrawal) and externalizing (e.g., aggression, self-abuse). However, a broad evaluation of behavioral abnormalities in a large cohort is lacking. OBJECTIVE: In this report, we describe profiles of internalizing and externalizing behaviors in individuals evaluated in the multi-center U.S. Rett Natural History Study. METHODS: Cross-sectional and longitudinal data were collected from 861 females with RTT and from 48 females who have MECP2 mutations without meeting criteria for RTT. Standard statistical methods including linear regression evaluated internalizing behavioral components from the Child Health Questionnaire (CHQ-PF50) and externalizing components from the Motor Behavioral Assessment (MBA). RESULTS: We found mildly to moderately severe internalizing behaviors in nearly all individuals with RTT, while externalizing behaviors were mild and uncommon. Internalizing behavior in RTT was comparable to groups with psychiatric disorders. Participants with mixed (internalizing and externalizing) behaviors were younger and less affected overall, but showed prominent self-injury and worsening internalizing behaviors over time. CONCLUSIONS: This study revealed that internalizing behaviors are common at a clinically significant level in RTT. Understanding clinical features associated with behavioral profiles could guide treatment strategies.