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Alcohol use disorder (AUD) is likely associated with complex transcriptional alterations in addiction-relevant brain regions. We characterize AUD-associated differences in cell type-specific gene expression and chromatin accessibility in the caudate nucleus by conducting a single-nucleus RNA-seq assay and a single-nucleus RNA-seq + ATAC-seq (multiome) assay on caudate tissue from 143 human postmortem brains (74 with AUD). We identified 17 cell types. AUD was associated with a higher proportion of microglia in an activated state and more astrocytes in a reactive state. There was widespread evidence for differentially expressed genes across cell types with the most identified in oligodendrocytes and astrocytes, including genes involved in immune response and synaptic regulation, many of which appeared to be regulated in part by JUND and OLIG2. Microglia-astrocyte communication via interleukin-1 beta, and microglia-astrocyte-oligodendrocyte interaction via transforming growth factor beta 1 were increased in individuals with AUD. Expression quantitative trait loci analysis revealed potential driver genes of AUD, including ADAL, that may protect against AUD in medium spiny neurons and interneurons. This work provides a thorough profile of the effects of AUD in the human brain and identifies several promising genes for further study.
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Tourette disorder (TD) is poorly understood, despite affecting 1/160 children. A lack of animal models possessing construct, face, and predictive validity hinders progress in the field. We used CRISPR/Cas9 genome editing to generate mice with mutations orthologous to human de novo variants in two high-confidence Tourette genes, CELSR3 and WWC1. Mice with human mutations in Celsr3 and Wwc1 exhibit cognitive and/or sensorimotor behavioral phenotypes consistent with TD. Sensorimotor gating deficits, as measured by acoustic prepulse inhibition, occur in both male and female Celsr3 TD models. Wwc1 mice show reduced prepulse inhibition only in females. Repetitive motor behaviors, common to Celsr3 mice and more pronounced in females, include vertical rearing and grooming. Sensorimotor gating deficits and rearing are attenuated by aripiprazole, a partial agonist at dopamine type II receptors. Unsupervised machine learning reveals numerous changes to spontaneous motor behavior and less predictable patterns of movement. Continuous fixed-ratio reinforcement shows that Celsr3 TD mice have enhanced motor responding and reward learning. Electrically evoked striatal dopamine release, tested in one model, is greater. Brain development is otherwise grossly normal without signs of striatal interneuron loss. Altogether, mice expressing human mutations in high-confidence TD genes exhibit face and predictive validity. Reduced prepulse inhibition and repetitive motor behaviors are core behavioral phenotypes and are responsive to aripiprazole. Enhanced reward learning and motor responding occur alongside greater evoked dopamine release. Phenotypes can also vary by sex and show stronger affection in females, an unexpected finding considering males are more frequently affected in TD.
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Dopamina , Mutación , Síndrome de Tourette , Animales , Síndrome de Tourette/genética , Síndrome de Tourette/fisiopatología , Síndrome de Tourette/metabolismo , Ratones , Femenino , Masculino , Humanos , Dopamina/metabolismo , Recompensa , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Aprendizaje/fisiología , Conducta Animal , Inhibición Prepulso/genética , Filtrado Sensorial/genéticaRESUMEN
Although genome-wide association studies (GWAS) have identified loci associated with alcohol consumption and alcohol use disorder (AUD), they do not identify which variants are functional. To approach this, we evaluated the impact of variants in 3' untranslated regions (3'-UTRs) of genes in loci associated with substance use and neurological disorders using a massively parallel reporter assay (MPRA) in neuroblastoma and microglia cells. Functionally impactful variants explained a higher proportion of heritability of alcohol traits than non-functional variants. We identified genes whose 3'UTR activities are associated with AUD and alcohol consumption by combining variant effects from MPRA with GWAS results. We examined their effects by evaluating gene expression after CRISPR inhibition of neuronal cells and stratifying brain tissue samples by MPRA-derived 3'-UTR activity. A pathway analysis of differentially expressed genes identified inflammation response pathways. These analyses suggest that variation in response to inflammation contributes to the propensity to increase alcohol consumption.
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Genome-wide association studies (GWAS) of electroencephalographic endophenotypes for alcohol use disorder (AUD) has identified noncoding polymorphisms within the KCNJ6 gene. KCNJ6 encodes GIRK2, a subunit of a G-protein-coupled inwardly rectifying potassium channel that regulates neuronal excitability. We studied the effect of upregulating KCNJ6 using an isogenic approach with human glutamatergic neurons derived from induced pluripotent stem cells (male and female donors). Using multielectrode arrays, population calcium imaging, single-cell patch-clamp electrophysiology, and mitochondrial stress tests, we find that elevated GIRK2 acts in concert with 7-21â d of ethanol exposure to inhibit neuronal activity, to counteract ethanol-induced increases in glutamate response, and to promote an increase intrinsic excitability. Furthermore, elevated GIRK2 prevented ethanol-induced changes in basal and activity-dependent mitochondrial respiration. These data support a role for GIRK2 in mitigating the effects of ethanol and a previously unknown connection to mitochondrial function in human glutamatergic neurons.
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Etanol , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Humanos , Masculino , Femenino , Estudio de Asociación del Genoma Completo , Neuronas , RespiraciónRESUMEN
Genome-wide association analysis (GWAS) of electroencephalographic endophenotypes for alcohol use disorder (AUD) has identified non-coding polymorphisms within the KCNJ6 gene. KCNJ6 encodes GIRK2, a subunit of a G protein-coupled inwardly-rectifying potassium channel that regulates neuronal excitability. How changes in GIRK2 affect human neuronal excitability and the response to repeated ethanol exposure is poorly understood. Here, we studied the effect of upregulating KCNJ6 using an isogenic approach with human glutamatergic neurons derived from induced pluripotent stem cells (male and female donors). Using multi-electrode-arrays, population calcium imaging, single-cell patch-clamp electrophysiology, and mitochondrial stress tests, we find that elevated GIRK2 acts in concert with 7-21 days of ethanol exposure to inhibit neuronal activity, to counteract ethanol-induced increases in glutamate response, and to promote an increase intrinsic excitability. Furthermore, elevated GIRK2 prevented ethanol-dependent changes in basal and activity-dependent mitochondrial respiration. These data support a role for GIRK2 in mitigating the effects of ethanol and a previously unknown connection to mitochondrial function in human glutamatergic neurons. SIGNIFICANCE STATEMENT: Alcohol use disorder (AUD) is a major health problem that has worsened since COVID, affecting over 100 million people worldwide. While it is known that heritability contributes to AUD, specific genes and their role in neuronal function remain poorly understood, especially in humans. In the current manuscript, we focused on the inwardly-rectifying potassium channel GIRK2, which has been identified in an AUD-endophenotype genome-wide association study. We used human excitatory neurons derived from healthy donors to study the impact of GIRK2 expression. Our results reveal that elevated GIRK2 counteracts ethanol-induced increases in glutamate response and intracellular calcium, as well as deficits in activity-dependent mitochondrial respiration. The role of GIRK2 in mitigating ethanol-induced hyper-glutamatergic and mitochondrial offers therapeutic promise for treating AUD.
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Tourette disorder (TD) is poorly understood, despite affecting 1/160 children. A lack of animal models possessing construct, face, and predictive validity hinders progress in the field. We used CRISPR/Cas9 genome editing to generate mice with mutations orthologous to human de novo variants in two high-confidence Tourette genes, CELSR3 and WWC1 . Mice with human mutations in Celsr3 and Wwc1 exhibit cognitive and/or sensorimotor behavioral phenotypes consistent with TD. Sensorimotor gating deficits, as measured by acoustic prepulse inhibition, occur in both male and female Celsr3 TD models. Wwc1 mice show reduced prepulse inhibition only in females. Repetitive motor behaviors, common to Celsr3 mice and more pronounced in females, include vertical rearing and grooming. Sensorimotor gating deficits and rearing are attenuated by aripiprazole, a partial agonist at dopamine type II receptors. Unsupervised machine learning reveals numerous changes to spontaneous motor behavior and less predictable patterns of movement. Continuous fixed-ratio reinforcement shows Celsr3 TD mice have enhanced motor responding and reward learning. Electrically evoked striatal dopamine release, tested in one model, is greater. Brain development is otherwise grossly normal without signs of striatal interneuron loss. Altogether, mice expressing human mutations in high-confidence TD genes exhibit face and predictive validity. Reduced prepulse inhibition and repetitive motor behaviors are core behavioral phenotypes and are responsive to aripiprazole. Enhanced reward learning and motor responding occurs alongside greater evoked dopamine release. Phenotypes can also vary by sex and show stronger affection in females, an unexpected finding considering males are more frequently affected in TD. Significance Statement: We generated mouse models that express mutations in high-confidence genes linked to Tourette disorder (TD). These models show sensorimotor and cognitive behavioral phenotypes resembling TD-like behaviors. Sensorimotor gating deficits and repetitive motor behaviors are attenuated by drugs that act on dopamine. Reward learning and striatal dopamine is enhanced. Brain development is grossly normal, including cortical layering and patterning of major axon tracts. Further, no signs of striatal interneuron loss are detected. Interestingly, behavioral phenotypes in affected females can be more pronounced than in males, despite male sex bias in the diagnosis of TD. These novel mouse models with construct, face, and predictive validity provide a new resource to study neural substrates that cause tics and related behavioral phenotypes in TD.
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Autism spectrum disorder (ASD), Tourette syndrome (TS), and attention-deficit/hyperactivity disorder (ADHD) display strong male sex bias, due to a combination of genetic and biological factors, as well as selective ascertainment. While the hemizygous nature of chromosome X (Chr X) in males has long been postulated as a key point of "male vulnerability", rare genetic variation on this chromosome has not been systematically characterized in large-scale whole exome sequencing studies of "idiopathic" ASD, TS, and ADHD. Here, we take advantage of informative recombinations in simplex ASD families to pinpoint risk-enriched regions on Chr X, within which rare maternally-inherited damaging variants carry substantial risk in males with ASD. We then apply a modified transmission disequilibrium test to 13,052 ASD probands and identify a novel high confidence ASD risk gene at exome-wide significance (MAGEC3). Finally, we observe that rare damaging variants within these risk regions carry similar effect sizes in males with TS or ADHD, further clarifying genetic mechanisms underlying male vulnerability in multiple neurodevelopmental disorders that can be exploited for systematic gene discovery.
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Trastorno por Déficit de Atención con Hiperactividad , Trastorno del Espectro Autista , Trastorno Autístico , Trastornos del Neurodesarrollo , Síndrome de Tourette , Humanos , Masculino , Femenino , Trastorno por Déficit de Atención con Hiperactividad/genética , Síndrome de Tourette/genética , Trastorno Autístico/genética , Trastorno del Espectro Autista/genéticaRESUMEN
Alcohol Use Disorder is a complex genetic disorder, involving genetic, neural, and environmental factors, and their interactions. The Collaborative Study on the Genetics of Alcoholism (COGA) has been investigating these factors and identified putative alcohol use disorder risk genes through genome-wide association studies. In this review, we describe advances made by COGA in elucidating the functional changes induced by alcohol use disorder risk genes using multimodal approaches with human cell lines and brain tissue. These studies involve investigating gene regulation in lymphoblastoid cells from COGA participants and in post-mortem brain tissues. High throughput reporter assays are being used to identify single nucleotide polymorphisms in which alternate alleles differ in driving gene expression. Specific single nucleotide polymorphisms (both coding or noncoding) have been modeled using induced pluripotent stem cells derived from COGA participants to evaluate the effects of genetic variants on transcriptomics, neuronal excitability, synaptic physiology, and the response to ethanol in human neurons from individuals with and without alcohol use disorder. We provide a perspective on future studies, such as using polygenic risk scores and populations of induced pluripotent stem cell-derived neurons to identify signaling pathways related with responses to alcohol. Starting with genes or loci associated with alcohol use disorder, COGA has demonstrated that integration of multimodal data within COGA participants and functional studies can reveal mechanisms linking genomic variants with alcohol use disorder, and potential targets for future treatments.
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Alcoholismo , Humanos , Alcoholismo/genética , Estudio de Asociación del Genoma Completo , Genómica , Consumo de Bebidas Alcohólicas , Etanol , Polimorfismo de Nucleótido SimpleRESUMEN
Recent genome-wide association studies (GWAS) have identified genetic markers of post-traumatic stress disorder (PTSD) in civilian and military populations. However, studies have yet to examine the genetics of PTSD while factoring in risk for alcohol dependence, which commonly co-occur. We examined genome-wide associations for DSM-IV PTSD among 4,978 trauma-exposed participants (31% with alcohol dependence, 50% female, 30% African ancestry) from the Collaborative Study on the Genetics of Alcoholism (COGA). We also examined associations of polygenic risk scores (PRS) derived from the Psychiatric Genomics Consortium (PGC)-PTSD Freeze 2 (N = 3533) and Million Veterans Program GWAS of PTSD (N = 5200) with PTSD and substance dependence in COGA, and moderating effects of sex and alcohol dependence. 7.3% of COGA participants met criteria for PTSD, with higher rates in females (10.1%) and those with alcohol dependence (12.3%). No independent loci met genome-wide significance in the PTSD meta-analysis of European (EA) and African ancestry (AA) participants. The PGC-PTSD PRS was associated with increased risk for PTSD (B = 0.126, p < 0.001), alcohol dependence (B = 0.231, p < 0.001), and cocaine dependence (B = 0.086, p < 0.01) in EA individuals. A significant interaction was observed, such that EA individuals with alcohol dependence and higher polygenic risk for PTSD were more likely to have PTSD (B = 0.090, p < 0.01) than those without alcohol dependence. These results further support the importance of examining substance dependence, specifically alcohol dependence, and PTSD together when investigating genetic influence on these disorders.
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Alcoholismo , Trastornos por Estrés Postraumático , Trastornos Relacionados con Sustancias , Humanos , Femenino , Masculino , Alcoholismo/genética , Trastornos por Estrés Postraumático/genética , Trastornos por Estrés Postraumático/psicología , Estudio de Asociación del Genoma Completo , Genómica , Trastornos Relacionados con Sustancias/genéticaRESUMEN
This review describes the genetic approaches and results from the family-based Collaborative Study on the Genetics of Alcoholism (COGA). COGA was designed during the linkage era to identify genes affecting the risk for alcohol use disorder (AUD) and related problems, and was among the first AUD-focused studies to subsequently adopt a genome-wide association (GWAS) approach. COGA's family-based structure, multimodal assessment with gold-standard clinical and neurophysiological data, and the availability of prospective longitudinal phenotyping continues to provide insights into the etiology of AUD and related disorders. These include investigations of genetic risk and trajectories of substance use and use disorders, phenome-wide association studies of loci of interest, and investigations of pleiotropy, social genomics, genetic nurture, and within-family comparisons. COGA is one of the few AUD genetics projects that includes a substantial number of participants of African ancestry. The sharing of data and biospecimens has been a cornerstone of the COGA project, and COGA is a key contributor to large-scale GWAS consortia. COGA's wealth of publicly available genetic and extensive phenotyping data continues to provide a unique and adaptable resource for our understanding of the genetic etiology of AUD and related traits.
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Alcoholismo , Humanos , Alcoholismo/genética , Estudio de Asociación del Genoma Completo , Estudios Prospectivos , Consumo de Bebidas Alcohólicas , FenotipoRESUMEN
BACKGROUND: Tourette syndrome (TS) is a childhood-onset neurodevelopmental disorder of complex genetic architecture and is characterized by multiple motor tics and at least one vocal tic persisting for more than 1 year. METHODS: We performed a genome-wide meta-analysis integrating a novel TS cohort with previously published data, resulting in a sample size of 6133 individuals with TS and 13,565 ancestry-matched control participants. RESULTS: We identified a genome-wide significant locus on chromosome 5q15. Integration of expression quantitative trait locus, Hi-C (high-throughput chromosome conformation capture), and genome-wide association study data implicated the NR2F1 gene and associated long noncoding RNAs within the 5q15 locus. Heritability partitioning identified statistically significant enrichment in brain tissue histone marks, while polygenic risk scoring of brain volume data identified statistically significant associations with right and left thalamus volumes and right putamen volume. CONCLUSIONS: Our work presents novel insights into the neurobiology of TS, thereby opening up new directions for future studies.
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Synonymous and noncoding single nucleotide polymorphisms (SNPs) in the KCNJ6 gene, encoding G protein-gated inwardly rectifying potassium channel subunit 2 (GIRK2), have been linked with increased electroencephalographic frontal theta event-related oscillations (ERO) in subjects diagnosed with alcohol use disorder (AUD). To identify molecular and cellular mechanisms while retaining the appropriate genetic background, we generated induced excitatory glutamatergic neurons (iN) from iPSCs derived from four AUD-diagnosed subjects with KCNJ6 variants ("Affected: AF") and four control subjects without variants ("Unaffected: UN"). Neurons were analyzed for changes in gene expression, morphology, excitability and physiological properties. Single-cell RNA sequencing suggests that KCNJ6 AF variant neurons have altered patterns of synaptic transmission and cell projection morphogenesis. Results confirm that AF neurons express lower levels of GIRK2, have greater neurite area, and elevated excitability. Interestingly, exposure to intoxicating concentrations of ethanol induces GIRK2 expression and reverses functional effects in AF neurons. Ectopic overexpression of GIRK2 alone mimics the effect of ethanol to normalize induced excitability. We conclude that KCNJ6 variants decrease GIRK2 expression and increase excitability and that this effect can be minimized or reduced with ethanol.
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Alcoholismo , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Humanos , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Etanol/farmacología , Etanol/metabolismo , Neuronas/metabolismo , Alcoholismo/genética , Alcoholismo/metabolismo , ElectroencefalografíaRESUMEN
Tourette syndrome (TS) is caused by multiple genetic and environmental factors. Yet, little is known about the interplay of these factors in the occurrence of tics. We investigated whether polygenic risk score (PRS) of TS and pregnancy-related factors together enhance the explained variance of tic occurrence in the Avon Longitudinal Study of Parents and Children (Ncases = 612; Ncontrols = 4,201; 50% male; mean age 13.8 years). We included a cumulative adverse pregnancy risk score, maternal anxiety and depression, and maternal smoking and alcohol use during pregnancy. We investigated possible joint effects of genetic and pregnancy-related risk factors using a multivariable approach, and explored mediation effects between the pregnancy-related risk factors in explaining tic presence. The PRS and the cumulative adverse pregnancy risk score, maternal anxiety, or maternal depression explained significantly more variance of tic presence compared to models including only the PRS. Furthermore, we found that the cumulative adverse pregnancy risk score mediated the association between several pregnancy-related factors (maternal anxiety, depression, and smoking) and tics. The combination of a PRS and pregnancy-related risk factors explained more variance of tics in a general population cohort compared to studying these factors in isolation.
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Tics , Síndrome de Tourette , Embarazo , Femenino , Humanos , Niño , Masculino , Adolescente , Tics/epidemiología , Tics/etiología , Estudios Longitudinales , Síndrome de Tourette/genética , Padres/psicología , Factores de RiesgoRESUMEN
Tourette syndrome (TS) is a neuropsychiatric disorder with involvement of genetic and environmental factors. We investigated genetic loci previously implicated in Tourette syndrome and associated disorders in interaction with pre- and perinatal adversity in relation to tic severity using a case-only (N = 518) design. We assessed 98 single-nucleotide polymorphisms (SNPs) selected from (I) top SNPs from genome-wide association studies (GWASs) of TS; (II) top SNPs from GWASs of obsessive-compulsive disorder (OCD), attention-deficit/hyperactivity disorder (ADHD), and autism spectrum disorder (ASD); (III) SNPs previously implicated in candidate-gene studies of TS; (IV) SNPs previously implicated in OCD or ASD; and (V) tagging SNPs in neurotransmitter-related candidate genes. Linear regression models were used to examine the main effects of the SNPs on tic severity, and the interaction effect of these SNPs with a cumulative pre- and perinatal adversity score. Replication was sought for SNPs that met the threshold of significance (after correcting for multiple testing) in a replication sample (N = 678). One SNP (rs7123010), previously implicated in a TS meta-analysis, was significantly related to higher tic severity. We found a gene-environment interaction for rs6539267, another top TS GWAS SNP. These findings were not independently replicated. Our study highlights the future potential of TS GWAS top hits in gene-environment studies.
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Trastorno por Déficit de Atención con Hiperactividad , Trastorno del Espectro Autista , Tics , Síndrome de Tourette , Trastorno por Déficit de Atención con Hiperactividad/genética , Trastorno del Espectro Autista/genética , Femenino , Interacción Gen-Ambiente , Estudio de Asociación del Genoma Completo , Humanos , Embarazo , Índice de Severidad de la EnfermedadRESUMEN
Tourette's Disorder (TD) is a neurodevelopmental disorder (NDD) that affects about 0.7% of the population and is one of the most heritable NDDs. Nevertheless, because of its polygenic nature and genetic heterogeneity, the genetic etiology of TD is not well understood. In this study, we combined the segregation information in 13 TD multiplex families with high-throughput sequencing and genotyping to identify genes associated with TD. Using whole-exome sequencing and genotyping array data, we identified both small and large genetic variants within the individuals. We then combined multiple types of evidence to prioritize candidate genes for TD, including variant segregation pattern, variant function prediction, candidate gene expression, protein-protein interaction network, candidate genes from previous studies, etc. From the 13 families, 71 strong candidate genes were identified, including both known genes for NDDs and novel genes, such as HtrA Serine Peptidase 3 (HTRA3), Cadherin-Related Family Member 1 (CDHR1), and Zinc Finger DHHC-Type Palmitoyltransferase 17 (ZDHHC17). The candidate genes are enriched in several Gene Ontology categories, such as dynein complex and synaptic membrane. Candidate genes and pathways identified in this study provide biological insight into TD etiology and potential targets for future studies.
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Síndrome de Tourette , Proteínas Relacionadas con las Cadherinas , Familia , Predisposición Genética a la Enfermedad/genética , Humanos , Proteínas del Tejido Nervioso/genética , Linaje , Serina Endopeptidasas , Síndrome de Tourette/genética , Secuenciación del ExomaRESUMEN
BACKGROUND: Genome-wide association studies (GWAS) of alcohol dependence (AD) and related phenotypes have identified multiple loci, but the functional variants underlying the loci have in most cases not been identified. Noncoding variants can influence phenotype by affecting gene expression; for example, variants in the 3' untranslated regions (3'UTR) can affect gene expression posttranscriptionally. METHODS: We adapted a high-throughput assay known as PASSPORT-seq (parallel assessment of polymorphisms in miRNA target sites by sequencing) to identify among variants associated with AD and related phenotypes those that cause differential expression in neuronal cell lines. Based upon meta-analyses of alcohol-related traits in African American and European Americans in the Collaborative Study on the Genetics of Alcoholism, we tested 296 single nucleotide polymorphisms (SNPs with meta-analysis p values ≤ 0.001) that were located in 3'UTRs. RESULTS: We identified 60 SNPs that affected gene expression (false discovery rate [FDR] < 0.05) in SH-SY5Y cells and 92 that affected expression in SK-N-BE(2) cells. Among these, 30 SNPs altered RNA levels in the same direction in both cell lines. Many of these SNPs reside in the binding sites of miRNAs and RNA-binding proteins and are expression quantitative trait loci of genes including KIF6,FRMD4A,CADM2,ADD2,PLK2, and GAS7. CONCLUSION: The SNPs identified in the PASSPORT-seq assay are functional variants that might affect the risk for AD and related phenotypes. Our study provides insights into gene regulation in AD and demonstrates the value of PASSPORT-seq as a tool to screen genetic variants in GWAS loci for one potential mechanism of action.
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Alcoholismo/genética , Polimorfismo de Nucleótido Simple/genética , Regiones no Traducidas 3'/genética , Línea Celular , Regulación de la Expresión Génica/genética , Estudios de Asociación Genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , HumanosAsunto(s)
COVID-19/epidemiología , Biología Computacional/educación , Proteasas 3C de Coronavirus , Curriculum , Educación a Distancia , Evolución Molecular , Pandemias , SARS-CoV-2 , Proteasas 3C de Coronavirus/química , Proteasas 3C de Coronavirus/genética , Humanos , SARS-CoV-2/química , SARS-CoV-2/genéticaRESUMEN
The OPRM1 A118G single nucleotide polymorphism (SNP rs1799971) gene variant encoding the N40D µ-opioid receptor (MOR) has been associated with dependence on opiates and other drugs of abuse but its mechanism is unknown. The frequency of G-allele carriers is ~40% in Asians, ~16% in Europeans, and ~3% in African-Americans. With opioid abuse-related deaths rising at unprecedented rates, understanding these mechanisms may provide a path to therapy. Here we generated homozygous N40D subject-specific induced inhibitory neuronal cells (iNs) from seven human-induced pluripotent stem (iPS) cell lines from subjects of European descent (both male and female) and probed the impact of N40D MOR regulation on synaptic transmission. We found that D40 iNs exhibit consistently stronger suppression (versus N40) of spontaneous inhibitory postsynaptic currents (sIPSCs) across multiple subjects. To mitigate the confounding effects of background genetic variation on neuronal function, the regulatory effects of MORs on synaptic transmission were recapitulated in two sets of independently engineered isogenic N40D iNs. In addition, we employed biochemical analysis and observed differential N-linked glycosylation of human MOR N40D. This study identifies neurophysiological and molecular differences between human MOR variants that may predict altered opioid responsivity and/or dependence in this subset of individuals.
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Neuronas/metabolismo , Trastornos Relacionados con Opioides/genética , Polimorfismo de Nucleótido Simple/genética , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Sinapsis/metabolismo , Línea Celular , Femenino , Humanos , Potenciales Postsinápticos Inhibidores , Masculino , Neuronas/citología , Receptores Opioides mu/químicaRESUMEN
Long interspersed elements-1 (LINE-1, L1) are retrotransposons that hold the capacity of self-propagation in the genome with potential mutagenic outcomes. How somatic cells restrict L1 activity and how this process becomes dysfunctional during aging and in cancer cells is poorly understood. L1s are enriched at lamin-associated domains, heterochromatic regions of the nuclear periphery. Whether this association is necessary for their repression has been elusive. Here we show that the sirtuin family member SIRT7 participates in the epigenetic transcriptional repression of L1 genome-wide in both mouse and human cells. SIRT7 depletion leads to increased L1 expression and retrotransposition. Mechanistically, we identify a novel interplay between SIRT7 and Lamin A/C in L1 repression. Our results demonstrate that SIRT7-mediated H3K18 deacetylation regulates L1 expression and promotes L1 association with elements of the nuclear lamina. The failure of such activity might contribute to the observed genome instability and compromised viability in SIRT7 knockout mice. Overall, our results reveal a novel function of SIRT7 on chromatin organization by mediating the anchoring of L1 to the nuclear envelope, and a new functional link of the nuclear lamina with transcriptional repression.
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Genoma , Lamina Tipo A/genética , Elementos de Nucleótido Esparcido Largo , Sirtuinas/genética , Transcripción Genética , Animales , Línea Celular , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Epigénesis Genética , Fibroblastos/citología , Fibroblastos/metabolismo , Heterocromatina/química , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Células K562 , Lamina Tipo A/metabolismo , Hígado/citología , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Miocardio/citología , Miocardio/metabolismo , Lámina Nuclear/metabolismo , Lámina Nuclear/ultraestructura , Sirtuinas/deficiencia , Sirtuinas/metabolismo , Testículo/citología , Testículo/metabolismoRESUMEN
BACKGROUND: Cystinuria is an inherited disorder of renal amino acid transport that causes recurrent nephrolithiasis and significant morbidity in humans. It has an incidence of 1 in 7000 worldwide making it one of the most common genetic disorders in man. We phenotypically characterized a mouse model of cystinuria type A resultant from knockout of Slc3a1. METHODS: Knockout of Slc3a1 at RNA and protein levels was evaluated using real-time quantitative PCR and immunofluorescence. Slc3a1 knockout mice were placed on normal or breeder chow diets and evaluated for cystine stone formation over time suing x-ray analysis, and the development of kidney injury by measuring injury biomarkers. Kidney injury was also evaluated via histologic analysis. Amino acid levels were measured in the blood of mice using high performance liquid chromatography. Liver glutathione levels were measured using a luminescent-based assay. RESULTS: We confirmed knockout of Slc3a1 at the RNA level, while Slc7a9 RNA representing the co-transporter was preserved. As expected, we observed bladder stone formation in Slc3a1-/- mice. Male Slc3a1-/- mice exhibited lower weights compared to Slc3a1+/+. Slc3a1-/- mice on a regular diet demonstrated elevated blood urea nitrogen (BUN) without elevation of serum creatinine. However, placing the knockout animals on a breeder chow diet, containing a higher cystine concentration, resulted in the development of elevation of both BUN and creatinine indicative of more severe chronic kidney disease. Histological examination revealed that these dietary effects resulted in worsened kidney tubular obstruction and interstitial inflammation as well as worsened bladder inflammation. Cystine is a precursor for the antioxidant molecule glutathione, so we evaluated glutathione levels in the livers of Slc3a1-/- mice. We found significantly lowered levels of both reduced and total glutathione in the knockout animals. CONCLUSIONS: Our results suggest that that diet can affect the development and progression of chronic kidney disease in an animal model of cystinuria, which may have important implications for patients with this disease. Additionally, reduced glutathione may predispose those with cystinuria to injury caused by oxidative stress. Word count: 327.