Unique functional responses differentially map onto genetic subtypes of dopamine neurons.

Dopamine neurons are characterized by their response to unexpected rewards, but they also fire during movement and aversive stimuli. Dopamine neuron diversity has been observed based on molecular expression profiles; however, whether different functions map onto such genetic subtypes remains unclear. In this study, we established that three genetic dopamine neuron subtypes within the substantia nigra pars compacta, characterized by the expression of Slc17a6 (Vglut2), Calb1 and Anxa1, each have a unique set of responses to rewards, aversive stimuli and accelerations and decelerations, and these signaling patterns are highly correlated between somas and axons within subtypes. Remarkably, reward responses were almost entirely absent in the Anxa1+ subtype, which instead displayed acceleration-correlated signaling. Our findings establish a connection between functional and genetic dopamine neuron subtypes and demonstrate that molecular expression patterns can serve as a common framework to dissect dopaminergic functions.

Comprehensive epigenomic profiling reveals the extent of disease-specific chromatin states and informs target discovery in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a common, highly heritable inflammatory arthritis characterized by enthesitis of the spine and sacroiliac joints. Genome-wide association studies (GWASs) have revealed more than 100 genetic associations whose functional effects remain largely unresolved. Here, we present a comprehensive transcriptomic and epigenomic map of disease-relevant blood immune cell subsets from AS patients and healthy controls. We find that, while CD14+ monocytes and CD4+ and CD8+ T cells show disease-specific differences at the RNA level, epigenomic differences are only apparent upon multi-omics integration. The latter reveals enrichment at disease-associated loci in monocytes. We link putative functional SNPs to genes using high-resolution Capture-C at 10 loci, including PTGER4 and ETS1, and show how disease-specific functional genomic data can be integrated with GWASs to enhance therapeutic target discovery. This study combines epigenetic and transcriptional analysis with GWASs to identify disease-relevant cell types and gene regulation of likely pathogenic relevance and prioritize drug targets.

Chromosome conformation capture approaches to investigate 3D genome architecture in Ankylosing Spondylitis.

Ankylosing Spondylitis (AS) is a chronic inflammatory arthritis of the spine exhibiting a strong genetic background. The mechanistic and functional understanding of the AS-associated genomic loci, identified with Genome Wide Association Studies (GWAS), remains challenging. Chromosome conformation capture (3C) and derivatives are recent techniques which are of great help in elucidating the spatial genome organization and of enormous support in uncover a mechanistic explanation for disease-associated genetic variants. The perturbation of three-dimensional (3D) genome hierarchy may lead to a plethora of human diseases, including rheumatological disorders. Here we illustrate the latest approaches and related findings on the field of genome organization, highlighting how the instability of 3D genome conformation may be among the causes of rheumatological disease phenotypes. We suggest a new perspective on the inclusive potential of a 3C approach to inform GWAS results in rheumatic diseases. 3D genome organization may ultimately lead to a more precise and comprehensive functional interpretation of AS association, which is the starting point for emerging and more specific therapies.

Regulatory T cells in spondyloarthropathies: genetic evidence, functional role, and therapeutic possibilities.

Regulatory T cells (Tregs) are a very specialized subset of T lymphocytes: their main function is controlling immune responses during inflammation. T-regs involvement in autoimmune and immune-mediated rheumatic diseases is well-described. Here, we critically review the up-to-date literature findings on the role of Tregs in spondyloarthropathies, particularly in ankylosing spondylitis (AS), a polygenic inflammatory rheumatic disease that preferentially affects the spine and the sacroiliac joints. Genetics discoveries helped in elucidating pathogenic T-regs gene modules and functional involvement. We highlight T-regs tissue specificity as crucial point, as T-regs might have a distinct epigenomic and molecular profiling depending on the different site of tissue inflammation. Furthermore, we speculate about possible therapeutic interventions targeting, or enhancing, Treg cells in spondyloarthropathies.

Perspectives on the Genetic Associations of Ankylosing Spondylitis.

Ankylosing spondylitis (AS) is a common form of inflammatory spinal arthritis with a complex polygenic aetiology. Genome-wide association studies have identified more than 100 loci, including some involved in antigen presentation (HLA-B27, ERAP1, and ERAP2), some in Th17 responses (IL6R, IL23R, TYK2, and STAT3), and others in macrophages and T-cells (IL7R, CSF2, RUNX3, and GPR65). Such observations have already helped identify potential new therapies targeting IL-17 and GM-CSF. Most AS genetic associations are not in protein-coding sequences but lie in intergenic regions where their direct relationship to particular genes is difficult to assess. They most likely reflect functional polymorphisms concerned with cell type-specific regulation of gene expression. Clarifying the nature of these associations should help to understand the pathogenic pathways involved in AS better and suggest potential cellular and molecular targets for drug therapy. However, even identifying the precise mechanisms behind the extremely strong HLA-B27 association with AS has so far proved elusive. Polygenic risk scores (using all the known genetic associations with AS) can be effective for the diagnosis of AS, particularly where there is a relatively high pre-test probability of AS. Genetic prediction of disease outcomes and response to biologics is not currently practicable.

The anxiety and ethanol intake controlling GAL5.1 enhancer is epigenetically modulated by, and controls preference for, high-fat diet.

Excess maternal fat intake and obesity increase offspring susceptibility to conditions such as chronic anxiety and substance abuse. We hypothesised that environmentally modulated DNA methylation changes (5mC/5hmC) in regulatory regions of the genome that modulate mood and consumptive behaviours could contribute to susceptibility to these conditions. We explored the effects of environmental factors on 5mC/5hmC levels within the GAL5.1 enhancer that controls anxiety-related behaviours and alcohol intake. We first observed that 5mC/5hmC levels within the GAL5.1 enhancer differed significantly in different parts of the brain. Moreover, we noted that early life stress had no significant effect of 5mC/5hmC levels within GAL5.1. In contrast, we identified that allowing access of pregnant mothers to high-fat diet (> 60% calories from fat) had a significant effect on 5mC/5hmC levels within GAL5.1 in hypothalamus and amygdala of resulting male offspring. Cell transfection-based studies using GAL5.1 reporter plasmids showed that 5mC has a significant repressive effect on GAL5.1 activity and its response to known stimuli, such as EGR1 transcription factor expression and PKC agonism. Intriguingly, CRISPR-driven disruption of GAL5.1 from the mouse genome, although having negligible effects on metabolism or general appetite, significantly decreased intake of high-fat diet suggesting that GAL5.1, in addition to being epigenetically modulated by high-fat diet, also actively contributes to the consumption of high-fat diet suggesting its involvement in an environmentally influenced regulatory loop. Furthermore, considering that GAL5.1 also controls alcohol preference and anxiety these studies may provide a first glimpse into an epigenetically controlled mechanism that links maternal high-fat diet with transgenerational susceptibility to alcohol abuse and anxiety.

CRISPR disruption and UK Biobank analysis of a highly conserved polymorphic enhancer suggests a role in male anxiety and ethanol intake.

Excessive alcohol intake is associated with 5.9% of global deaths. However, this figure is especially acute in men such that 7.6% of deaths can be attributed to alcohol intake. Previous studies identified a significant interaction between genotypes of the galanin (GAL) gene with anxiety and alcohol abuse in different male populations but were unable to define a mechanism. To address these issues the current study analysed the human UK Biobank cohort and identified a significant interaction (n = 115,865; p = 0.0007) between allelic variation (GG or CA genotypes) in the highly conserved human GAL5.1 enhancer, alcohol intake (AUDIT questionnaire scores) and anxiety in men. Critically, disruption of GAL5.1 in mice using CRISPR genome editing significantly reduced GAL expression in the amygdala and hypothalamus whilst producing a corresponding reduction in ethanol intake in KO mice. Intriguingly, we also found the evidence of reduced anxiety-like behaviour in male GAL5.1KO animals mirroring that seen in humans from our UK Biobank studies. Using bioinformatic analysis and co-transfection studies we further identified the EGR1 transcription factor, that is co-expressed with GAL in amygdala and hypothalamus, as being important in the protein kinase C (PKC) supported activity of the GG genotype of GAL5.1 but less so in the CA genotype. Our unique study uses a novel combination of human association analysis, CRISPR genome editing in mice, animal behavioural analysis and cell culture studies to identify a highly conserved regulatory mechanism linking anxiety and alcohol intake that might contribute to increased susceptibility to anxiety and alcohol abuse in men.

Disruption of c-MYC Binding and Chromosomal Looping Involving Genetic Variants Associated With Ankylosing Spondylitis Upstream of the RUNX3 Promoter.

Background: Ankylosing Spondylitis (AS) is a common form of inflammatory spinal arthritis with a complex aetiology and high heritability, involving more than 100 genetic associations. These include several AS-associated single nucleotide polymorphisms (SNPs) upstream of RUNX3, which encodes the multifunctional RUNT-related transcription factor (TF) 3. The lead associated SNP rs6600247 (p = 2.6 × 10-15) lies ∼13kb upstream of the RUNX3 promoter adjacent to a c-MYC TF binding-site. The effect of rs6600247 genotype on DNA binding and chromosome looping were investigated by electrophoretic mobility gel shift assays (EMSA), Western blotting-EMSA (WEMSA) and Chromosome Conformation Capture (3C). Results: Interrogation of ENCODE published data showed open chromatin in the region overlapping rs6600247 in primary human CD14+ monocytes, in contrast to the Jurkat T cell line or primary human T-cells. The rs6600247 AS-risk allele is predicted to specifically disrupt a c-MYC binding-site. Using a 50bp DNA probe spanning rs6600247 we consistently observed reduced binding to the AS-risk "C" allele of both purified c-MYC protein and nuclear extracts (NE) from monocyte-like U937 cells. WEMSA on U937 NE and purified c-MYC protein confirmed these differences (n = 3; p < 0.05). 3C experiments demonstrated negligible interaction between the region encompassing rs6600247 and the RUNX3 promoter. A stronger interaction frequency was demonstrated between the RUNX3 promoter and the previously characterised AS-associated SNP rs4648889. Conclusion: The lead SNP rs6600247, located in an enhancer-like region upstream of the RUNX3 promoter, modulates c-MYC binding. However, the region encompassing rs6600247 has rather limited physical interaction with the promoter of RUNX3. In contrast a clear chromatin looping event between the region encompassing rs4648889 and the RUNX3 promoter was observed. These data provide further evidence for complexity in the regulatory elements upstream of the RUNX3 promoter and the involvement of RUNX3 transcriptional regulation in AS.

The IL-17/IL-23 Axis and Its Genetic Contribution to Psoriatic Arthritis.

Psoriatic arthritis (PsA) is a chronic inflammatory disease belonging to the family of spondyloarthropathies (SpA). PsA commonly aggravates psoriasis of the skin and frequently manifests as an oligoarthritis with axial skeletal involvement and extraarticular manifestations including dactylitis, enthesitis, and uveitis. The weight of genetic predisposition to psoriasis and PsA is illustrated by the concordance rates in monozygotic twins which clearly demonstrate that genomics is insufficient to induce the clinical phenotype. The association of PsA with several single nucleotide polymorphisms (SNPs) at the IL23R locus and the involvement of Th17 cells in the immunopathogenesis of PsA clearly put the IL-23/IL-17 axis in the spotlight. The IL-23 and IL-17 cytokines have a pivotal role in the chronic inflammation of the synovium in PsA and are also prominent in the skin lesions of those with PsA. In this review, we focus on the genetic association of the IL-23/IL-17 axis with PsA and the contribution of these master cytokines in the pathophysiology of the disease, highlighting the main cell types incriminated in PsA and their specific role in the peripheral blood, lesional skin and joints of patients. We then provide an overview of the approved biologic drugs targeting the IL-23/IL-17 axis and discuss the advantages of genetic stratification to enhance personalized therapies in PsA.