Affective states are typically accompanied by facial expressions, but these behavioral manifestations are highly variable. Even highly arousing and negative valent experiences, such as pain, show great instability in facial affect encoding. The present study investigated which neural mechanisms are associated with variations in facial affect encoding by focusing on facial encoding of sustained pain experiences. Facial expressions, pain ratings, and brain activity (BOLD-fMRI) during tonic heat pain were recorded in 27 healthy participants. We analyzed facial expressions by using the Facial Action Coding System (FACS) and examined brain activations during epochs of painful stimulation that were accompanied by facial expressions of pain. Epochs of facial expressions of pain were coupled with activity increase in motor areas  as well as in areas involved in nociceptive processing, including primary and secondary somatosensory cortex, posterior and anterior insula, and the anterior part of the mid-cingulate cortex. In contrast, prefrontal structures  were less activated during incidences of facial expressions, consistent with a role in down-regulating facial displays. These results indicate that incidences of facial encoding of pain reflect activity within nociceptive pathways interacting or possibly competing with prefrontal inhibitory systems that gate the level of expressiveness. With the growing impact of the affective sciences  and recorded facial expressions and brain activation  to investigate neural mechanisms associated with incidences of facial displays of pain.Twenty-seven, healthy volunteers  aged 18 to 30 years  participated in the study and were recruited via advertisements posted on the campuses of the Université de Montréal and McGill University. All participants were included in a previously published report on a phasic pain paradigm . The contact probe was attached to the left lateral lower leg. Baseline temperature was always set to 38 °C. To ensure that temperature intensities were perceived as painful but tolerable in all participants (to prevent floor as well as ceiling effects), temperature intensities were tailored to the individual pain threshold. Following a familiarization trial, heat pain thresholds were determined by using the method of adjustment in a mock MRI scanner immediately before the scanning session . Participants were brought into the scanner room for the experiment immediately after.Pain was induced experimentally by an MRI-compatible, Peltier-based, thermal stimulator with a 3 × 3 cmTonic heat stimuli were administered according to protocol of the Tonic Heat Pain Model  and converted linearly to values between 0 and 100. The VAS for sensory intensity of pain was labeled with verbal anchors from “no pain” (0) to “extremely strong pain” (100). Pain unpleasantness was labeled with “no pain” (0) to “extremely unpleasant pain” (100). All participants were instructed about the conceptual distinction between sensory intensity of pain and pain unpleasantness following the instructions of Price et al. . VAS senDuring both functional scans, the face of the subject was videotaped using a small MRI-compatible camera  mounted onto the head coil. The camera was carefully positioned to capture the face of the subject reflected through a mirror attached above the head coil, without blocking the visual field of the subject. The onset of each thermal stimulus was marked automatically on the video recording by using a signal sent from the stimulator to the sound card. A software designed for the analysis of observational data  was used to segment the videos and to score facial expressions into a time-related database. Time segments of 2 minutes beginning with stimulus reaching the target temperature were selected for scoring of facial expressions. We quantified facial expressions using the Facial Action Coding System . Structural images were acquired by using a high-resolution, T1-weighted MP-RAGE sequence .Imaging data were acquired at the “Unité de Neuroimagerie Fonctionnelle” of the “Centre de recherche de l’Institut de gériatrie de Montréal” using a 3T Siemens Magneton TIM Trio magnetic resonance imaging (MRI) system with a 12-channel head coil. Participants were placed in a comfortable position and their head stabilized with foam pads and headphones. Earplugs were also given to reduce the noise from the scanner. The two functional runs were preceded by one high-resolution, anatomical scan of 9 minutes. A total of 110 whole-brain volumes were acquired during each functional scan using blood oxygen level-dependent (BOLD) contrast. Each functional volume comprised 40 interleaved axial slices of 3.40-mm thickness parallel to the AC-PC line (in-plane resolution 3.44 x 3.44 mm). Volumes were acquired by using a gradient echo, echo-planar (EPI) T2*-weighted sequence , executed in Matlab 8.6 . Functional images were first pre-processed with slice-time correction, and motion corrected by realigning all images to the first image using six-parameter rigid body transformation and reslicing with fourth-degree B-spline interpolation. Additionally, images were preprocessed with SPM’s extension toolbox ArtRepair (v.5b3) to detect and repair by interpolation, outlier volumes before coregistraton and spatial normalization  in SPM12 to identify brain regions showing significant changes in BOLD signal  were included in the design matrix as nuisance regressors. Additionally, mean signals across voxels from the white matter and the cerebrospinal fluid were added as covariates of no interest to remove possible physiological noise.t-test. Significance was determined using the False Discovery Rate method applied at the voxel level (FDR q = 0.05).Contrast images showing areas of the brain associated with epochs of facial expressions of pain during tonic pain were generated for each subject and were subsequently used in second-level analyses to compute group average map by using one-sample The mean (±SD) pain threshold was 45.58 °C (± 0.85). The tonic stimulation (target intensity +1.3 °C above threshold) was rated with an average VAS-intensity score of 78.3 (± 10.7) and an average VAS-unpleasantness score of 83.9 (± 9.9).During the 480 seconds of painful stimulation, facial expressions of pain were displayed on average across 142.1 seconds. Thus, approximately 30% of the sustained pain experience was accompanied by facial encoding of pain. Breaking the occurrence of facial encoding of pain down into discrete epochs, we found that participants on average displayed 26 epochs of facial expressions of pain across the 480 seconds of painful stimulation; each epoch lasted on average for 5.36 seconds . Examples of facial encoding of pain occurring during the tonic pain stimulation as well as the lack thereof are shown in Fig. Positive brain activations significantly associated with epochs of facial encoding of pain during tonic pain stimulation Fig. A FDR q ; Fig. 3BIn contrast, peaks of significant decrease in BOLD (FDR q = 0.05) were observed in the frontal areas ranging from dorsolateral to ventrolateral and medial prefrontal areas Table ; Fig. 3CThe purpose of the present study was to investigate which neural mechanisms may underly variations in facial affect encoding by focusing on facial expressions during tonic pain stimulation. The data presented were obtained using a subsample of a study where we assessed facial responses to phasic heat pain  and premotor areas, conforming with previous findings Bair . BesidesThe association between S1 activity and the occurrence of facial expressions of pain may reflect spontaneous fluctuations in the intensity of the pain experienced (independently of any change in the noxious input). This interpretation derives from several previous studies demonstrating that changes within S1 are closely associated with changes in the subjective pain intensity (Bushnell et al., The aMCC represents a particularly interesting area in the context of tonic stimulation where participants are instructed to tolerate moderate-to-strong pain for several minutes. In addition to the nociceptive input received from the spinal cord through the mediodorsal nucleus, the aMCC is involved in regulating motor and behavioral responses (Dum et al., Facial expressions of affective states are under the control of complex regulatory processes. The present results replicate our previous findings showing that facial encoding of pain is accompanied by a decrease in prefrontal activation (Kunz et al., The regulation of facial expressions is believed to be socially learned. Facial expressions of pain are rather stimulus-driven and reflexive during the first months of life, and children gradually learn to regulate their facial expressions according to social display rules (Larachotte et al., In summary, the neural activation and deactivation found in association with facial encoding of pain imply that spontaneous fluctuations in nociceptive activity and endogenous motivational factors may interact or even compete with prefrontal inhibitory systems to regulate facial expressions.In the present study, facial expression and neural activity were assessed continuously across tonic stimulation, whereas self-report was only assessed at the end of each 2-minute stimulus. Thus, we cannot determine to which degree facial encoding of pain was linked to changes in pain intensity ratings across tonic stimulation. However, given the low associations found between facial and subjective responses (Kunz et al., Even the experience of moderate-to-strong pain intensities does not lead to a constant visible facial display of pain. In incidences where pain was facially displayed, we found heightened thalamo-cortical nociceptive processing in combination with a deactivation of prefrontal structures. This implies that incidences of facial displays of pain reflect nociceptive activity and possibly endogenous motivational factors interacting or even competing with prefrontal inhibitory systems.


AUTS2 gene results in the upregulation of this gene in neurons. Overall, our study provides pertinent TAD boundary evolutionary conservation annotations and showcases the functional importance of TAD evolution.Topological associating domains (TADs) are self-interacting genomic units crucial for shaping gene regulation patterns. Despite their importance, the extent of their evolutionary conservation and its functional implications remain largely unknown. In this study, we generate Hi-C and ChIP-seq data and compare TAD organization across four primate and four rodent species and characterize the genetic and epigenetic properties of TAD boundaries in correspondence to their evolutionary conservation. We find 14% of all human TAD boundaries to be shared among all eight species (ultraconserved), while 15% are human-specific. Ultraconserved TAD boundaries have stronger insulation strength, CTCF binding, and enrichment of older retrotransposons compared to species-specific boundaries. CRISPR-Cas9 knockouts of an ultraconserved boundary in a mouse model lead to tissue-specific gene expression changes and morphological phenotypes. Deletion of a human-specific boundary near the autism-related  The authors show that the deletion of ultraconserved TAD boundaries affects gene expression and phenotype, highlighting TAD evolution’s function. Human-specific TAD boundaries reveal a role in brain development and disease. Chromatin conformation capture assays, namely Hi-C, have shed light on 3D genome organization and revealed that chromosomes are compartmentalized into kilo- to mega-base scale segments termed topologically associated domains (TADs). TADs often contain gene(s) that can interact with regulatory elements located in the same domain, while interactions across domains are prevented by flanking elements that are often bound by specific proteins including CTCF, cohesin complexes and RNA-polymerase II3. These insulating elements are commonly known as TAD boundaries, and considering their significant role in defining functional interaction domains, they are thought to be critical for maintaining proper genome function.The three-dimensional (3D) organization of the genome plays a fundamental role in orchestrating chromatin interactions that regulate gene expression and shape phenotypes4, limb malformation5 and neurodevelopmental disease6. As a result, the function and position of TAD boundaries in the genome are likely subject to some degree of evolutionary constraint and conservation. When TADs were first described9, a direct comparison between human and mouse TAD organization revealed that a large portion of TAD boundaries was shared between the two species 7. This finding was corroborated by several subsequent lines of direct and indirect evidence. For example, direct comparisons of the Hox10 and Six11 loci detected similar TAD structures across several species. Further convincing—albeit indirect—evidence of TAD conservation came from studies providing evidence for selection against structural variations (SVs) that disrupt genome topology; In the human population, evolutionary SVs were shown to be depleted at TAD boundaries, while this pattern was absent in patients with developmental delay12. In addition, in the highly rearranged gibbon genome13, breakpoints of evolutionary chromosomal rearrangements between human and gibbon were found to be enriched at TAD boundaries, resulting in the preservation of TADs even when synteny is lost14. Findings from these and other studies have reinforced the notion that TAD boundaries remain unchanged during evolution due to deleterious consequences of TAD disruption15. However, most of the evidence supporting TAD conservation is indirect and/or only limited to a few species or loci. Moreover, a few recent studies present evidence refuting high conservation of TAD boundaries across species, even those as closely related as human and chimpanzee16. Hence, there is growing uncertainty around the level of evolutionary TAD conservation17 and a need for further reassessment of TAD conservation via comprehensive direct cross-species comparisons.Disruption of TAD boundaries has been associated with ectopic gene interactions, gene misregulation and aberrant phenotypes such as cancer18. Therefore, a comparative investigation of TAD boundaries and the gene regulatory domains they form can shed new light on mechanisms contributing to adaptations, speciation, and evolution. Regions of high TAD conservation represent crucial loci whose disruption may lead to reduced fitness19, while regions with evolutionarily diverged TAD organization may be associated with gene interaction/expression changes that underlie evolutionary novelties across species. Indeed, differences in 3D genomic interactions in human-chimpanzee induced pluripotent stem cells (iPSC) have been associated with inter-specific differences in gene expression16. Furthermore, 3D chromatin structures identified in the fetal human cortex, but not in rhesus and mouse, may have contributed to human-specific features of brain development20, highlighting the power of using a comparative approach in assessing TAD evolution and inferring TAD boundary functionality.Gene regulation plays a key role in species evolutionHere, in order to assess the level of evolutionary TAD conservation in mammals, we identified TAD boundaries across four primate and four rodent species and performed direct comparisons to group them based on their evolutionary conservation. Characterization of the genetic and epigenetic profiles of TAD boundary across evolutionary groups, as well as generation of in vivo and in vitro deletions, allowed us to investigate the function of these boundaries and their relevance to development, disease and evolution.13. The two selected gibbon species have very different karyotypes and chromosome numbers , referred to as Nomascus and Hylobates hereon. From rodents, we selected mouse and rat, as well as two relatively closely related Mus species with divergent karyotypes , referred to as Caroli and Pahari from hereon21. We used public Hi-C data for human lymphoblastoid cell line (LCL) (Supplementary Data r = 0.66–0.75)  maps Fig. . The selN = 6542) and rhesus (N = 7845) having the lowest and highest numbers of predicted TADs, respectively  TADs across the genomes of all species, with mouse N = 654 and rhes22. To validate that the effect of tissue origin is weaker than the evolutionary relationship between samples, we investigated patterns of cross-species/tissue TAD boundary overlap and distribution. We found that boundaries were shared more often among closely related species, even when originating from different tissues . We observed that as the level of conservation increased, the insulation scores decreased, indicating that boundaries shared by more species lead to higher separation between neighboring TADs   of the boundaries in the human genome in LCL were ultraconserved, 6% (N = 491) were primate-conserved, and 15% (N = 1130) were human-specific. A roughly similar distribution was found in the mouse genome in liver, where 19.1% (N = 1023) of the TAD boundaries were ultraconserved, 2.1% (N = 115) were rodent-conserved, and 15.0% (N = 807) were mouse-specific (Supplementary Data We then used the phylogenetic relationship between species to classify human and mouse TAD boundaries based on their evolutionary conservation and estimated age into the following groups: (1) ultraconserved boundaries  (Supplementary Note p < 0.001)  Fig. . Hence, 25 and as such, their co-option may have contributed to the evolution of TAD boundaries27. Analysis of CTCF binding sites located inside TAD boundaries in each species showed that over half of them overlap TEs across species  of SINE and DNA repeat classes at CTCF binding sites inside ultraconserved and primate-conserved boundaries  at CTCF binding sites of ultraconserved, rodent-conserved and mouse-specific boundaries. Similar to the human genome, among SINEs, the MIR family was significantly over-represented in CTCF binding sites of ultraconserved TAD boundaries, suggesting that this TE family has been involved in the evolution of TAD organization at least since the common ancestor of primates and rodents. Of note, in the mouse genome, the rodent-specific B1 and B2 repeat families were enriched at CTCF binding sites across all evolutionary boundary groups  can introduce CTCF binding sites upon their insertion in host genomesies Fig. . Among tent Fig. . In content Fig. , which iups Fig. , support32 have shown that breakpoints of evolutionary chromosomal rearrangements are over-represented at TAD boundaries, and this has been interpreted as evidence that selection pressures maintain TAD integrity even when genome synteny is disrupted. Here, we took advantage of our high-resolution Hi-C data and improved genome assemblies33  and the lowest number of BOS was found in Caroli  were species-specific and likely occurred independently in the two genera (Supplementary Data p < 0.05) . We observed an over-representation of species-specific and low-conservation TAD boundaries near BOS Fig. . Next, w05) Fig.  in both 15. As such, there may be an association between the TAD boundary conservation level and the nature and function of nearby genes. Specifically, we expect ultraconserved TAD boundaries to be associated with genes whose disruption interferes with fundamental biological functions. Consistently, using the Genomic Regions Enrichment of Annotations Tool (GREAT36) to investigate ontology terms associated with TAD boundaries (“Methods”) revealed significant enrichment of genes implicated in ‘anatomical structure formation during embryogenesis’ nearby ultraconserved boundaries in the human genome  that were validated via PCR, sequencing and Southern blot, and used two of them for downstream phenotyping  showed no apparent overt cardiac defects, including no differences in cardiac wall thickness between the left and right ventricles . However, LV walls in B5234−/− mice appeared more compact with less extracellular tissue/space in between cardiac cells and displayed irregular trabeculation in some regions   Fig. .Fig. 4HeMeis2’s role in ear development37, we also characterized the morphology of the utricle and the cochlea in neonatal mice . Hair cells appeared to have normal morphology and bundle orientation in both the cochlea and utricle  and schizophrenia42. Human-specific boundaries were also associated with several genes implicated in brain disease and development, such as the Autism Susceptibility Candidate 2 (AUTS2) gene, a key regulator of transcriptional networks and a mediator of epigenetic regulation in neurodevelopment. AUTS2 is implicated in ASD and other neurological diseases44 and contains the most significantly accelerated genomic region differentiating humans from other primates43. To further investigate the association of human-specific TAD boundary deletions with human disease, we tested their overlap with pathological copy number variants (CNVs), previously identified from 29,085 children with developmental delay and 19,584 healthy controls45. Using permutation analysis (Supplementary Note n = 335 out of 1130 human-specific boundaries) compared to healthy controls (n = 161) .The emergence of new TAD boundaries changes local TAD organization, which could lead to the evolution of novel and adaptive gene regulatory modules. To this end, among the different boundary conservation groups, the human-specific boundaries are of particular significance as they may be relevant to the evolution of human-specific traits and adaptations. When investigating gene ontologies (GO) associated with human-specific TAD boundaries, we detected a significant enrichment of pathways pertaining to ‘positive regulation of synapse assembly’ . The deletion was performed in induced pluripotent stem cells (iPSC) WTC11-Ngn2 cell line, which allows quick and robust differentiation to neurons46  and validated them via PCR , while no changes were detected in expression of the HPRT1 housekeeping gene  in human neurons located ~200Kb upstream of s46 Fig. . We obtaBB3 Fig. . We founene Fig. . We alsocus Fig. . Altoget7, challenging the long-standing notion that TAD organization is highly conserved across tissues and species. It should be noted however, that we used relatively strict criteria for inferring conservations by requiring boundaries to successfully lift over to the human reference genome and only considered boundaries as overlapping if they were within 10 Kb of each other. Furthermore, our cross-species Hi-C datasets originated from two different cell types, LCL and liver, and thus any boundary identified as ultraconserved had to be developmentally stable between these two tissues, adding constraints to our classification criteria. Nonetheless, our data highlights that boundaries in a genome vary in their level of evolutionary conservation, and this variation is associated with overall differences in their genetic and epigenetic properties. For example, we observed that ultraconserved TAD boundaries have higher insulation strength and harbor more CTCF binding sites compared to species-specific boundaries. Contributing to this pattern may be the fact that boundaries that have lower insulation strength and fewer CTCF binding sites are more likely to be missed during boundary annotation, resulting in arbitrarily lower conservation estimates. Nevertheless, these findings are in line with a previous study that reported higher CTCF binding site clustering and stronger CTCF affinity at TAD boundaries shared across five murine species, compared to boundaries with lower conservation47. Moreover, CTCF binding sites at ultraconserved boundaries overlap with older transposable elements (namely MIRs), whereas human-specific boundaries are associated with more recent families (ERVs), reinforcing the notion that different waves of transposable elements have been shaping genome topology through the insertion of CTCF binding sites at different evolutionary times30. Altogether, this indicates that recurrent emergence and maintenance of several, possibly redundant, CTCF binding sites contribute to the higher conservation and insulation strength of ultraconserved TAD boundaries.In this study, we investigated mammalian TAD conservation by obtaining and comparing high-resolution Hi-C and ChIP-seq data across four primate and four rodent species Fig. . We esti48, and their disruption can lead to gene misregulation and aberrant phenotypes49. Hence, it has been proposed that TAD boundaries evolve under evolutionary constraints that prevent their disruption even in the face of evolutionary chromosomal rearrangements14. In support of this hypothesis, previous studies have shown a co-localization of evolutionary breaks of synteny (BOS) and TAD boundaries, even in species with highly rearranged genomes, like gibbons51. Here, we also report significant co-localization between BOS and TAD boundaries in the gibbon species, which carry highly rearranged genomes13. Additionally, our evolutionary classification enabled us to detect over-representation of species-specific  TAD boundaries among those that co-localize with BOS, across all species and within each gibbon genome  in vivo deletions of TAD boundaries near developmental genes in mice observed severe phenotypes when two boundaries classified as ultraconserved in our dataset were deleted. Of note, the severity of phenotypes reported in this study seems to broadly correlate with the deletion size and number of CTCF clusters removed. As our deletion was smaller in size and removed just one CTCF binding site, it is possible that retention of functionally redundant neighboring CTCF binding sites mitigated the effects of the deletion.An important implication of the functional connection between TAD boundaries and genes is that selection pressures under which TAD boundaries evolve are likely influenced by the function of nearby genes. Consistently, in the human genome, we observed that genes associated with ultraconserved boundaries were enriched in pathways pertaining to anatomical structure formation during embryogenesis, whose disruption will likely lead to developmental defects. Moreover, manipulating an ultraconserved TAD boundary in a mouse model altered interactions and expression of a neighboring gene and resulted in putatively maladaptive heart phenotypes. While we did not detect acute phenotypes, a recent study41. For instance, the timing of synaptic development and expression of relevant genes is prolonged in humans compared to macaque and chimpanzee, and synapse density is highly increased in humans in comparison to other primates52. Mounting evidence also places synapse assembly and its pathology at the heart of several human cognitive disorders, such as ASD and schizophrenia42, suggesting that disruption of human-specific boundaries might also be a contributing factor in human neuropathology. It should be noted, however, that this study used Hi-C data from liver and LCL tissues to identify human-specific boundaries, and given that TAD organization can vary across tissues22, the association of human-specific boundaries with brain-related genes and pathways will likely be stronger if investigated directly in brain cells22. Nevertheless, we found significantly more overlap between human-specific TAD boundaries and CNVs identified in children with developmental delay45, compared to healthy controls. This is in line with recent work that found TAD-shuffling to alter enhancer-promoter interactions and be associated with a variety of human developmental disorders53. We additionally showed that deletion of a candidate human-specific TAD boundary upstream of the AUTS2 gene resulted in differences in genomic interaction patterns  and gene expression at the AUTS2 locus in iPSC-derived human neurons, providing evidence for the functionality of human-specific boundaries in human brain cells.We also identified TAD boundaries exclusively present in the human genome and showed that they were significantly associated with genes implicated in synapse assembly. Of note, the human brain undergoes unique synapse patterning, which is thought to contribute to its extraordinary complexity and connectivityIn summary, we generated high-resolution Hi-C and ChIP-seq data from several species and used direct multi-species comparison to stratify TAD boundaries in the mouse and human genomes based on evolutionary conservation. Our study highlights the remarkable range of diversity that exists in the evolutionary conservation of TAD boundaries, regardless of species (mouse vs. human) and tissue (LCL vs. liver). Furthermore, by comparing genetic and epigenetic features of boundaries across conservation groups and investigating the function of candidate TAD boundaries via in vivo and in vitro manipulations, we provided new insights into the functional implications of TAD boundary evolution. While our study underlines the importance of TAD conservation in maintaining proper gene regulation patterns during evolution, it also showcases how recent changes in TAD organization can contribute to the emergence of evolutionary novelties and species-specific traits. Future comprehensive comparative studies in additional tissues and developmental stages will help us better understand the contributions of TAD organization to biological function, as well as if and how disrupting these structures can lead to pathology.Sex effects were not considered in the study design.54. Alignments were converted to a multi-resolution Hi-C matrix using pairtools (https://github.com/mirnylab/pairtools). Hi-C data between biological replicates were combined after verifying their correlation based on the Pearson correlation as calculated by the HiCExplorer package55 . Thus, TAD boundaries are annotated by identifying statistically significant local minima in insulation score curves.We used liver tissue and lymphoblastoid cell lines (LCLs) to generate Hi-C libraries with the Arima Hi-C Kit following the manufacturer’s protocol. Briefly, ~10 mg frozen pellets of the homogenized fixed liver or LCL were lysed and conditioned before chromatin digestion. The digested chromatin was then filled in and biotinylated before ligation. Next, chromatin was protein-digested and reverse crosslinking overnight, followed by purification. The purified DNA was then sonicated using the bioruptor pico (Diagenode) and size selected before library preparation using the NEB DNA Ultra II, following Arima’s protocol. The Hi-C DNA was bound to streptavidin beads before enzymatic end prep, adapter ligation, DNA release by heat incubation, and lastly, PCR to barcode and amplify the libraries. Libraries were sequenced on the Illumina HiSeq2500 or NovaSeq6000. Raw Hi-C sequencing was processed using the HiCUP pipelineTo perform cross-species comparisons and determine TAD boundary evolutionary conservation, we lifted TAD boundaries identified in all species to the hg38 coordinates, generated a union boundary map and used paradigms of boundary presence/absence across species to stratify union boundaries into groups, as detailed in the Supplementary Note 57, to determine the proportion of boundaries overlapping with genes. We used the TEanalysis tool58 to examine TE enrichment at CTCF ChIP-seq peaks  within TAD boundaries in each conservation group.We compared insulation score, chromatin structure, CTCF binding sites, and overlap with genes and transposable elements (TE) across TAD boundary conservation groups in human and mouse genomes. Boundary insulation scores were generated as part of the TAD boundary annotation analysis and compared using the Wilcoxon signed-rank test. CTCF binding sites and chromatin states were determined by using a combination of public and newly generated H3K4me1, H3K27ac, K3K4me3, H3K27me3 and CTCF ChIP-seq data from a female and a male of each of the eight species examined in this study (Supplementary Data https://github.com/carbonelab/lastz-pipeline), followed by alignment chaining and filtering using UCSC tools56. For Nomascus, we took advantage of an improved version of the genome assembly (Asia_NLE_v1) based on PacBio CLR and guided by Nleu3 generated by the Eichler lab (Supplementary Note https://github.com/carbonelab/axtToSyn), which elongates alignment blocks that are longer than 1Kb and have a minimum alignment score of 100,000 by merging them with other blocks on the same chromosome and strand. Elongated alignment blocks represent synteny blocks between two genomes, thus synteny breakpoints were defined as 1Kb regions flanking each elongated synteny block in the target genome. To transfer coordinates of synteny breakpoints from the target genome coordinates to those of the query genome, we used BLAT59 with the following parameters: -stepSize=5 -repMatch=2253 -minScore=20 -minIdentity=0. The BLAT results were manually evaluated and if the BLAT score of the second highest-scoring hit was within 10% of the top-scoring hit, the breakpoint was annotated as duplicated and removed. Breakpoints that survived this filtration step were further manually inspected to remove BOS overlapping segmental duplications and large repeats. Only curated breakpoints that survived both filtration steps were used for downstream analysis  of evolutionary chromosomal rearrangements using a custom pipeline in which all pairwise comparisons of known chromosomes of the “target” genome are aligned to the “query” genome using LASTZ (https://github.com/carbonelab/hicpileup) we visualized aggregate Hi-C contact frequencies in a 2 Mb window centered at synteny breakpoints. The median insulation score was visualized in 600 Mb windows centered at synteny breakpoints. Since BOS are often found in GC-rich regions, we used stratified random sampling (based on %GC content) to generate a random set of BOS with similar GC content. We used Fisher’s exact test to compare the observed number of TAD boundary overlapping BOS (at least 1 bp)  targeted an 18Kb region  including an ultraconserved boundary near the Meis2 gene using i-GONAD60 described in Supplementary Note All transgenic animal experiments were conducted on FVB/NJ  mice in accordance with the Guide for the Care and Use of Laboratory Animals established by the National Institutes of Health. Protocols were approved by the Institutional Animal Care and Use Committees (IACUC) at UCSF. All mice were allowed ad libitum access to food and water and were maintained on a 12 h light/dark cycle in a climate-controlled facility at 40–60% humidity and at room temperature. Our CRISPR deletion B234−/− ta46 . Briefly, WTC11-ngn2 cells were cultured in mTeSR1 media (STEMCELL Technologies) with daily media changes following normal WTC11 maintenance protocols. Cells were seeded at a density of 300k cells per 6-well in mTeSR1 media plus Rock Inhibitor (Selleckchem) and cultured for one day. WTC11-ngn2 p37 ± 21 cells  were then transfected with 800 ng of each of the four sgRNAs (Supplementary Data B14804−/− and wild-type cells using AllPrep DNA/RNA Mini kit (Qiagen). cDNA was synthesized from extracted RNA using SuperScript™ III Reverse Transcriptase (Invitrogen) following the manufacturer’s protocol. cDNA was diluted 1:10 and used for qPCR with SsoFast EvaGreen Supermix (BioRad). qPCR reactions were done in triplicate and normalized against Gapdh. Frozen B14804−/− and B14804+/+ neurons were used to generate Hi-C libraries using the Arima Hi-C Kit as described earlier (n = 2 per genotype). Hi-C libraries were sequenced on the Illumina NovaSeq6000 platform. Raw Hi-C data was merged across replicates and interaction matrices and insulation scores were calculated as described above  that included the human-specific B14804 boundary in WTC11-ngn2 cellsFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Supplementary Data 11Reporting SummarySource Data


Neutrophil responses are critical during inflammatory and infective events, and neutrophil dysregulation has been associated with poor patient outcomes. Immunometabolism is a rapidly growing field that has provided insights into cellular functions in health and disease. Neutrophils are highly glycolytic when activated, with inhibition of glycolysis associated with functional deficits. There is currently very limited data available assessing metabolism in neutrophils. Extracellular flux (XF) analysis assesses real time oxygen consumption and the rate of proton efflux in cells. This technology allows for the automated addition of inhibitors and stimulants to visualise the effect on metabolism. We describe optimised protocols for an XFe96 XF Analyser to (i) probe glycolysis in neutrophils under basal and stimulated conditions, (ii) probe phorbol 12-myristate 13-acetate induced oxidative burst, and (iii) highlight challenges of using XF technology to examine mitochondrial function in neutrophils. We provide an overview of how to analyze XF data and identify pitfalls of probing neutrophil metabolism with XF analysis. In summary we describe robust methods for assessing glycolysis and oxidative burst in human neutrophils and discuss the challenges around using this technique to assess mitochondrial respiration. XF technology is a powerful platform with a user-friendly interface and data analysis templates, however we suggest caution when assessing neutrophil mitochondrial respiration. XF technology allows automated addition of different stimuli or inhibitors with flexibility over timing to visualise the effect of stimulation and inhibitory factors on cellular metabolism. XF technology requires low cell numbers relative to other forms of respirometry and allows for simultaneous measurement of PER and OCR as indicators for glycolysis and mitochondrial function, respectively. The streamlined workflow combined with user-friendly interface and analysis templates have seen a worldwide boom in the use of these instruments for probing cellular metabolism. In this manuscript we describe optimised protocols for using an XFe96 XF Analyser to probe (i) glycolysis in neutrophils under basal and stimulated conditions and (ii) phorbol 12-myristate 130-acetate (PMA)-induced oxidative burst. We highlight limitations of using XF technology to examine mitochondrial function in neutrophils. Finally, we provide an overview of how to analyze XF data and identify pitfalls of probing neutrophil metabolism with XF analysis.11 otherwise healthy volunteers aged 22-49 years old were recruited from staff members in the Queen Elizabeth Hospital Birmingham. Written informed consent was given by all participants in accordance with the University of Birmingham Research Ethics Committee (ERN 12-1185R2).Neutrophils are notoriously challenging cells to work with due to their short lifespan and propensity for activation. The upmost care must be taken during cell isolation and assays should be commenced immediately following isolation to reduce risk of apoptosis.g for 20 minutes with no brake or acceleration. Neutrophils were removed and washed in phosphate-buffered saline  (PBS)  before being resuspended at 4 x 106/mL in phenol red and sodium bicarbonate free, filter sterilized Roswell Park Memorial Institute-1640 (RPMI) , supplemented with 2mM L-glutamine, 1mM sodium pyruvate, 25mM glucose and 5mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) , pH 7.4, which will be referred to as XF media. Neutrophil purity was >99% for all assays, except where neutrophil purity is detailed in the Figure legend.Peripheral blood neutrophils were isolated from lithium-heparin anticoagulated  whole venous blood as previously described . Briefly® Cell-Tak™  solution and stored overnight at 4°C. On the day of the assay pre-coated microplates were bought to room temperature and seeded with 2 x 105 cells/well  or 5 x 104 cells/well (oxidative burst) in 50µL XF media. Wells A1, A12, H1 and H12 contained only 50 µL/well XF media to account for background correction wells. The XFe96 microplate was centrifuged at 200 x g for 1 minute with maximum acceleration but no brake at room temperature before incubation in a non-CO2, 37°C incubator for 25-30 minutes. Wells were observed under a light microscope to ensure cell adherence before gently adding 130 µL/well pre-warmed XF media, after addition of media the plate was incubated for a further 15 minutes in a non-CO2, 37°C incubator. 25 µL of each inhibitor/stimulant was added to injection ports A-C of the sensor cartridge  as follows, with stated concentrations referring to final well concentrations: Glycolysis- port A: 160 nM PMA , port B: 2 µg/ml oligomycin   or XF media, port C: compound of interest. N-Formyl methionyl-leucyl-phenylalanine (fMLP) . Heat killed Streptococcus pneumoniae (SP) and non-typeable Haemophillus influenzae (NTHI) were donated by Dr Kylie Belchamber  coated with 22.4 µg/mL Corningigomycin   was used to convert ECAR to PER using the buffer factor 2.8 as calculated by the manufacturer’s protocol  for RPMI supplemented with 25 mM glucose. Glycolytic parameters were calculated as follows: Basal glycolysis - PER value prior to the first injection, PMA-induced glycolysis - maximum PER reached over the course of the assay, and glycolytic response to PMA – PMA-induced glycolysis minus basal glycolysis. Area under the curve analysis was performed on GraphPad Prism to generate oxidative burst parameters. Mitochondrial respiration parameters were calculated by importing OCR data into pre-formatted analysis templates in Microsoft Excel to calculate basal respiration, ATP-linked respiration, proton leak respiration, maximal respiration, and non-mitochondrial respiration. Mean and standard error of the mean (SEM) are presented for data. Paired t-test was performed for data 4A-B. Linear regression analysis was performed in GraphPad Prism for mitochondrial data in 8 A-D.OCR and extracellular acidification rate (ECAR data) were exported from WAVE to GraphPad Prism and Microsoft Excel for downstream analysis. Seahorse analytics  measured by XF technology can be used to quantify oxidative burst response in mononuclear cells such as neutrophils. We highlight this in the representative oxidative burst trace in 4 and 5 x 105 neutrophils/well were seeded onto XFe96 cell culture microplates as described in methods. Baseline OCR was measured over a period of 4 measurement cycles  prior to sequential injections of 2 µM rotenone plus antimycin A and 160 nM PMA to establish oxidative burst test parameters  three minutes following injection  and mtDAMPs (p= 0.002) compared to unprimed neutrophils. Peak oxygen consumption is rapid within three minutes of injection and overall oxygen consumption is lower than that induced by PMA. Peak oxygen consumption induced by fMLP was 38.12 pmol O mtDAMPs Figure 42 generated in the tricarboxylic acid cycle can be spontaneously or enzymatically hydrated to carbonic acid (H2CO3), which dissociates to + in media at physiological pH. Of significance, conversion of one glucose molecule to lactate yields 2 lactate + 2 H+, whereas complete oxidation of one glucose to CO2 yields 6 +, therefore when extracellular acidification of glucose is oxidized to CO2, extracellular acidification is three times higher than conversion to lactate. Moreover, XF analysis of ECAR from changes in media pH does not consider the buffering capacity of the media, which often contain low amounts of HEPES or other buffering agents. It is therefore also important to correct for buffering capacity of the XF assay media. This correction converts ECAR into proton efflux rate (PER), which is preferable when interpreting glycolytic flux.XF analysis can also provide detailed information on cellular glycolytic flux. Until recently , ECAR waGlycolytic rate assessed using XF technology is obtained in cells exposed to exogenous glucose and mitochondrial complex inhibitors such as oligomycin or rotenone plus antimycin A. Typically, treatment of cells with mitochondrial respiratory complex inhibitors such as oligomycin increases PER because of increased glycolytic flux to sustain cellular ATP supply to meet the energetic needs of the cell when mitochondrial ATP synthesis is abolished. However, in neutrophils, under basal conditions with surplus exogenous glucose, oligomycin has no effect on PER Figure 55 isolated neutrophils per well. As indicated by increases in PER, basal glycolytic rate positively correlated with increased cell density , OCR is below the limit of detection for the XFe96 analyser and thus OCR obtained from neutrophils at these densities should be treated with caution when inferring mitochondrial respiratory activity.To establish optimal neutrophil seeding density for assessment of mitochondrial respiration using XF analysis, a range of cell seeding densities were examined from 0.5 – 5 x 10min/well Figure 7hreshold  is not a5 cells/well) make it difficult to obtain reliable OCR  pmol O2/min/well at 50% purity  seeded at 2 x 10Neutrophils are highly glycolytic cells ; inhibit4 neutrophils/well, we saw a reduction in the size of the oxidative burst response. Typically, it would be anticipated that greater cell seeding densities would produce a larger oxidative burst response, however, due to cell overcrowding and/or reduced PMA stimulation relative to number of cells, this was not the case. We demonstrated that 5 x 104 neutrophils/well combined with an injection strategy incorporating 2 µM rotenone plus 2 µM antimycin A followed by 160 nM PMA, produces a substantial and consistent oxidative burst response, without maximising the neutrophil reactive oxygen species (ROS) response. This allows for detection of both elevated and suppressed oxidative burst responses caused by disease states or exogenous treatments. We show that PMA concentrations lower than 160 nM do not produce a consistent peak following PMA injection, and therefore area under the curve analysis is unable to quantify a reliable oxidative burst response. Data in SP, NTHI, or LPS did not induce a detectable increase in oxygen consumption with or without priming. Heat killed bacteria and LPS have been used by others to induce oxidative burst, although with different methods (Activated neutrophils rapidly consume oxygen as reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase forms superoxide anions, reactive oxygen species and hydrogen peroxide, a process commonly referred to as oxidative burst. PMA is a protein kinase C activator and a robust activator of neutrophils, albeit non-physiological . In cell methods , 28.More commonly used methods to measure oxidative burst detect generation of ROS by a range of fluorescent, chemiluminescent probes rather than detecting environmental oxygen consumption . While tvia mitochondrial respiration and therefore upregulate glycolysis to meet the energetic demands of the cell. When testing this assay on human neutrophils, we observed no change in glycolysis after oligomycin injection. A recent publication has highlighted that by inhibiting mitochondrial respiration using mitochondrial respiratory complex inhibitors in resting state cells, cells only shift to glycolysis as a compensatory mechanism for basal ATP demand requirements, thus these agents may not actually shift cells to maximum glycolytic capacity as previously thought  are no longer accepted as representative for glycolysis. Media often has a large buffering capacity, which by its nature can mask pH changes, and subsequent acidification rates. Therefore, by calculating and applying a buffering factor for each specific media to account for the buffering capacity, ECAR values can be converted to PER, a more representative readout for glycolytic tests .XF analyzers account for the pH and oxygen content in background wells, which contain no cells. This enables background correction of the values to ensure readouts represent actual metabolism rather than changes in ambient OXF analyzers allow for high throughput data and multiple metabolic tests to be performed on a single assay plate, with a minimal demand for cell numbers. The dynamics of oxygen consumption and change in pH over the duration of the assay allows for the depiction of cellular metabolism and metabolic responses over time. XF analyzers accurately predict lactate production compared to direct lactate measurement . Other mXF analyzers are highly sensitive; however, care must be taken in ensuring an evenly distributed monolayer of cells is formed. If cells are not distributed directly under the probe, or if they begin to detach from the base of the well due to disruptions, probes can lack fine discrimination. Further, introduction of pharmaceutical treatments or other stimulants/inhibitors requires further assay validation to ensure they do not interact with the probes themselves, which are sensitive to pH. Despite these limitations, we have demonstrated how XF technology can be harnessed for neutrophil bioenergetic analysis. XF analyzers are costly to run compared to other respirometers, however the high throughput and automated nature of the system allows for efficient data collection.In conclusion, we have presented optimised protocols for the assessment of neutrophil glycolysis and oxidative burst using XF technology. The metabolic tests outlined here can be applied to any project context by incorporating pharmacological treatments, disease state neutrophils or neutrophils from different developmental stages. We demonstrate robust methodology to achieve neutrophil monolayers to perform metabolic assays for assessing glycolytic rate and oxidative burst. We highlight the challenges of these assays and mitigating strategies when assessing mitochondrial respiration. Overall, XF technology represents a powerful platform with a user-friendly interface and data analysis templates which has the potential to revolutionise our understanding of neutrophil metabolism. The protocols described in our manuscript provide a template to enhance discovery and ensure a standardised approach to neutrophil measurements using XF analysis.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by University of Birmingham Research Ethics Committee (ERN 12-1185R2). The patients/participants provided their written informed consent to participate in this study.AJ and FG performed all experiments. JB, AS, AJ and FG designed the study with critical review by DT, ES and CM. Data analysis was undertaken by AJ, FG, and JB. All authors contributed to the article and approved the submitted version.


Genomic imprinting disorders are caused by the disruption of genomic imprinting processes leading to a deficit or increase of an active allele. Their unique molecular mechanisms underlying imprinted genes offer an opportunity to investigate epigenetic-based therapy for reactivation of an inactive allele or reduction of an active allele. Current treatments are based on managing symptoms, not targeting the molecular mechanisms underlying imprinting disorders. Here, we highlight molecular approaches of therapeutic candidates in preclinical and clinical studies for individual imprinting disorders. These include the significant progress of discovery and testing of small molecules, antisense oligonucleotides, and CRISPR mediated genome editing approaches as new therapeutic strategies. We discuss the significant challenges of translating these promising therapies from the preclinical stage to the clinic, especially for genome editing based approaches. Igf2r) was the first imprinted gene discovered in mice in 1991 [Genomic imprinting is a special form of epigenetic regulation, resulting in monoallelic gene expression depending on parent-of-origin. Genomic imprinting was first described in the early 1980s in mice from the elegant pronuclear transfer experiments by Surani and Solter . They sh in 1991 . In huma in 1991 –6. Sever in 1991 –10. Firs in 1991 –14. Thir in 1991 , 16. Fou in 1991 . These f in 1991 , 22.The disruption of imprinting processes during gametogenesis and the expression of imprinted genes causes significant developmental defects and diseases in humans referred to as genomic imprinting disorders , 9, 23. Prader–Willi (PWS) and Angelman syndrome (AS) are the first examples of genomic imprinting disorders described in the late 1980s –34. CurrDNMT3A), DNA demethyltransferase (TET3), H1 linker histone, histone modifying enzymes, and chromatin remodelers  have been implicated in ASD and NDD [Recent large scale genomic studies have uncovered a list of genes that encode proteins of epigenetic machinery implicated in neurodevelopmental disorders (NDD) specifically for ASD –40. For  and NDD –45. It rLike most other genetic disorders, there are no effective molecular treatments for imprinting disorders. Current treatments for imprinting disorders are symptom-based interventions and often ineffective. The molecular mechanism underlying imprinting disorders is primarily due to loss of active alleles or duplication of repressed or inactive alleles via different genetic mechanisms , 12, 46.Epigenetic-based therapy has been investigated extensively in cancers and mostly used the pharmacological approach –51. The The molecular basis of epigenetic based therapy for imprinting disorders is to unsilence/reactivate the expression of disease-causing genes from the repressed allele in imprinted loci via pharmacological or molecular genetic manipulation. The basic concept of epigenetic-based therapy has been intensely studied in cancers for more than two decades mostly by pharmacological approach –51. ManyEpigenetic modifications can be achieved by small molecules or other pharmacological interventions altering the function of epigenetic machinery directly associated with transcriptional regulation of imprinted genes. Histone modifying enzymes are common targets for development of novel therapies for human diseases, primarily for cancers. Many small molecules targeting histone deacetylases (HDACs) have been approved by the FDA and are in clinical trials and or used as therapies for cancers and other diseases . These iUbe3a gene, Philpot and Roth groups have screened >3000 small molecules [Ube3a gene from the repressed paternal chromosome in neurons both in vitro and in vivo. One of these topoisomerase inhibitors is topotecan, a FDA approved drug for treatment of metastatic cancers. Intervention with topotecan in the AS Ube3a maternal deficiency mouse model rescues the neurobehavioral phenotypes. The significant toxicity associated with topotecan has precluded it from being considered further in human AS. In a mouse model for PWS, EHMT2/G9a inhibitors are found as effective small molecules to unsilence the expression of normal repressed PWS candidate genes from the maternal chromosome 15q11-q13 region in both human PWS derived cells and PWS mouse model [Kcnk9 allele with an increase of H3K27 acetylation in Kcnk9 promoter and intronic regions [Peg13, a differentially methylated region located upstream of Kcnk9 gene [To systemically identify new epigenetic drugs, high content small molecule screenings have been carried out to examine the feasibility of reactivating the repressed imprinted genes using proper markers in cell-based assays . Screeniolecules . This scse model . The bio regions . This innk9 gene . These sASOs are short, synthetic, single-stranded oligodeoxynucleotides that bind to target pre-mRNAs . ChemicaUBE3A gene is subject to brain specific imprinting [UBE3A gene is exclusively expressed from the maternal chromosome in neurons [UBE3A gene remains fully characterized. It has been demonstrated in rodent models that the maternal and neuron specific expression of Ube3a is mediated by the expression of a paternally expressed long non-coding antisense RNA to Ube3a (Ube3a-ATS) [Ube3a-ATS by ASO and CRISPR based gene editing at the DNA and RNA levels unsilence the expression of sense Ube3a in brains [Ube3a by ASO is capable of rescuing the neurobehavioral and neurophysiological impairments in AS maternal Ube3a deficiency mouse model [As a therapeutic strategy, ASOs have been introduced and extensively tested for over two decades to improve their activity in clinical trials . With teprinting , 70. The neurons . The exae3a-ATS) , 72. Inhn brains –76. The se model , 77–79. se model .UBE3A-ATS. However, due to the transient nature of ASO treatment after entry into cells, repeated intrathecal injections are necessary to maintain clinical efficacy if it eventually becomes a standard therapy. The requirement of sedation for intrathecal administration of ASOs poses significant medical and psychosocial stress to AS children and families. CRISPR/Cas9 gene editing offers an attractive alternative to ASO. Two recent studies have demonstrated that Cas9 mediated gene editing can inactivate the expression of Ube3a-ATS and reactivate the expression of Ube3a from the paternal chromosome in vitro and in vivo using an adeno-associated virus (AAV) or lentivirus delivery method [Ube3a-ATS rescues the neurobehavioral phenotypes in the AS Ube3a maternal deficiency mouse model. These studies provide initial evidence supporting the feasibility of using Cas9 editing to treat AS. Like CRISPR/Cas9 mediated gene editing in other genetic diseases, safety concerns related to virus delivery methods and potential off-target effects due to Cas9 editing remain to be evaluated thoroughly before moving to human trials. Conceptually, an unbiased CRISPR based screening could be designed to screen for a genetic locus that could unsilence imprinted genes and lead to the development of a new treatment. This has not been reported in literature so far.The studies of ASOs provide a proof of concept to support molecular therapy by manipulating y method , 75. A s6A), increases RNA stability and translation efficiency, leading to enhancement of gene expression [6A demethylase, dCasRx-ALKBH5 shows bidirectional modulation depending on targeting mRNA [UBE3A-ATS has been reported to reactivate the expression of UBE3A from the paternal chromosome and rescue some neurobehaviors in mice [Non-coding RNAs are frequently associated with imprinted gene clusters and strongly implicated in the regulation of imprinted clusters , 80–82. pression . CRISPR-pression . As a m6ing mRNA . The tec in mice . HoweverCRISPR-based epigenome editing technologies have been developed to enable manipulation of the epigenome and regulate expression of targeted genes –94. For 5-methylcytosine (5mC) in CG dinucleotides (CpG) plays a critical role in imprinting establishment during development –99. CpG FMR1 (Fragile X Syndrome) in cells and in vivo [Snrpn gene in a rodent model [MECP2 promoter for its reactivation from inactivated Xi chromosome in Rett syndrome human embryonic stem cells and derived neurons [As an enzymatic eraser, the ten-eleven translocation dioxygenase family of genes (TET1-3) encodes enzymes that oxidize 5mC to 5-hydroxymethylcytosine (5hmC) leading to active DNA demethylation and increase of gene transcription in general. dCas9-TET1/TET3 targeting to methylated CpG island contributes to specific gene activation in various disease models , 107–109 in vivo , the CpGnt model , and the neurons . With thManipulating posttranslational modifications of histones in in vitro and in vivo experimental systems using CRISPR/dCas9-mediate editing have shown a strong effect on gene regulation bidirectionally as well as specificity. For example, dCas9-p300 and dCas9-dMSK1 induce an increase of target gene transcription through acetylation and phosphorylation to specific residues on histones, respectively , 112  or transcriptional activation (CRISPRa) systems have been actively developed due to eagerness for efficacy improvement. Various advanced versions of these systems can recruit multiple transcription factors to control gene transcription. More than two kinds of catalytic domains are fused with dCas9 for synergetic effects on target gene regulation , 96. Beycis and trans by tethering to reach distal regulatory elements [For epigenetic gene silencing, dCas9 is fused with Krüppel associated box (KRAB), derived from zinc finger domain, leading to decrease of chromatin accessibility and high H3K9me3 levels on target regulatory regions with recruitment of other repressors . An advaelements . This toSNORD116 is considered to be the critical gene, where a deficiency is responsible for key PWS clinical features [SNURF-SNRPN gene [SNRPN from the maternal chromosome in cells derived from a patient with PWS due to a paternal deletion of 15q11-q13 [Snrpn promoter region including CpG islands, suggesting a potent alternative tool [AS and PWS are caused by deficiency of maternally and paternally expressed genes on chromosome 15q11-q13, respectively. De novo ~6 Mb paternal deletion of the 15q11-q13 region is found in ~70% of PWS patients, followed by maternal UPD ~27%), and rare imprinting center mutations  have a de novo ~6 Mb maternal deletion including the utations –139. UBE3 region . The exp% have a of Snrpn , 72. Theof Snrpn . Topotecse model . However neurons , 142. Bose model –75, 143.se model . AdditioWith the FDA approved ASO in phase 1/2a trial for AS, the prospect of developing epigenetic molecular therapies for other genomic imprinting disorders is encouraging. The number of genomic imprinting disorders and the populations affected are relatively small. However, because of the unique molecular defects associated with genomic imprinting disorders and molecular mechanisms underlying imprinting regulations, genomic imprinting disorders remain the best opportunity for a proof of principle study of developing epigenetic therapy for genetic diseases. The lessons learned and tools developed are immediately applicable to other non-imprinting genetic disorders. For example, genetic defects that lead to haploinsufficiency of single genes are found in 10–15% of cases with ASD and even higher in other NDD. The approach of upregulating the expression of normal alleles by epigenome editing is an attractive avenue for developing molecular treatments. Despite these promises, challenges remain significant. For pharmacological based epigenetic therapy, the broad effects of epigenetic modulating drugs remain a potential concern for clinical applications particularly for mild or moderate presentation of the disease. It is possible that side effects are dose dependent and likely cell and tissue type specific. The careful assessment of these issues may ease the concern from FDA regulatory concerns. For ASO and gene editing based therapies, the efficiency of delivery platforms remains to be improved especially for the brain as a target organ. For example, the repeat dosing for ASO intrathecal delivery used in AS clinical trials is not optimal for patient care. The virus delivery platform for Cas9 editing has its own inherited and well recognized limitations. New non-viral delivery platforms have been developed in recent years but they are suboptimal for brain disorders. Lastly, for any gene editing approach, the genome wide off-target events of Cas9/gRNA remain a concern conceptually. The challenge is to assess the impact of off-target events technically and physiologically, and how to define an acceptable threshold of genome wide off-target events from a regulatory and safety perspective. Nevertheless, it is reasonable to predict that more promising breakthroughs utilizing CRISPR based genome and epigenome editing will emerge as an effective treatment modality in the near future.


IL6R, IL1β or β-tubulin gene isoforms, respectively, that were significantly associated with serum C-reactive protein (CRP) in a large European genome-wide association study . These were paired with summary statistics from a large GWAS of AS in European patients  to then perform primary inverse-variance weighted random effect and sensitivity MR analyses for each exposure. This analysis showed that genetically proxied tocilizumab was associated with reduced risk of AS . Genetically proxied canakinumab was not associated with risk of AS , and only one suitable SNP was identified to proxy the effect of colchicine . The finding that genetically proxied tocilizumab was associated with reduced risk of AS is concordant with an inflammatory hypothesis of AS pathogenesis. Inhibition of IL6R may be a promising therapeutic target for AS management.There are currently no approved pharmacological treatment options for aortic stenosis (AS), and there are limited identified drug targets for this chronic condition. It remains unclear whether inflammation plays a role in AS pathogenesis and whether immunomodulation could become a therapeutic target. We evaluated the potentially causal association between inflammation and AS by investigating the genetically proxied effects of tocilizumab , canakinumab (IL1β inhibitor) and colchicine (β-tubulin inhibitor) through a Mendelian randomisation (MR) approach. Genetic proxies for these drugs were identified as single nucleotide polymorphisms (SNPs) in the gene, enhancer or promoter regions of  If left untreated, severe AS can lead to up to 50% mortality within two years of symptom onset2. Transcatheter and surgical valve replacement are currently the mainstays of treatment4, with a limited role for medical therapies. The 2020 American guidelines for management of valvular heart disease recognised a role for treatment of dyslipidemia and hypertension, particularly with angiotensin-converting enzyme inhibitors or angiotensin receptor blockers8. Similarly the 2021 European guidelines advised treatment of coexistent hypertension, although these guidelines also importantly note that medical therapies have not yet been shown to alter the natural history of AS4. There is consequently a need to better understand the causal mechanisms underlying AS to facilitate targeted preventative and therapeutic medications.The prevalence of aortic stenosis (AS) is increasing with the ageing population. Approximately 12% of individuals over age 75 years are diagnosed with AS, of whom over a quarter have severe AS12. Positron emission tomography concordantly demonstrates increased tracer uptake in patients with AS versus normal valves13. A genome-wide association study (GWAS) meta-analysis further identified the IL6 locus as significantly associated with AS14 and observational studies have demonstrated an association between increased IL6 protein levels and AS16. However, whether inflammation is a cause, consequence or correlate of AS remains unclear. Several studies have shown similarities in the pathogenesis between AS and coronary atherosclerosis, and several randomised trials have demonstrated the effectiveness of the anti-inflammatory medications canakinumab17 and colchicine19 for coronary heart disease. It is therefore hypothesised that immunomodulatory drugs, such as tocilizumab , canakinumab (IL1β inhibitor) or colchicine (β-tubulin inhibitor) may prove effective for the management of AS.Several modalities of evidence suggest that inflammation plays a role in the development of AS. Immunohistochemical analysis of surgical and autopsy aortic valves has demonstrated elevated expression of inflammatory cell adhesion molecules and cytokines, and increased inflammatory cell infiltration in valves with AS compared to normal valves20. This approach has previously been used to demonstrate significant associations between both raised systolic blood pressure and low-density lipoprotein cholesterol with risk of not only coronary disease21 but also AS7. MR relies on the premise that a proportion of an individual’s phenotype is determined by genetic polymorphisms that are randomly inherited at birth. The random inheritance of phenotype-determining genetic polymorphisms is analogous to assignment to a treatment group in a randomised control trial22. This randomisation process can overcome several issues with observational studies including reverse causation and confounding, and may therefore facilitate causal inferences when clinical trials are unavailable.Mendelian randomisation (MR) offers an opportunity to investigate the potentially causal relationship between risk factors of interest, such as inflammation, and outcomes, such as ASIL6R gene. This approach has been used in MR analyses to demonstrate that genetically proxied IL6R inhibition is significantly associated with reduced risk of coronary artery disease and ischemic stroke25. Moreover, it has previously been shown that genetically proxied IL6R inhibition, estimated using a SNP in the IL6R gene, is associated with a similar biochemical effect as administering tocilizumab26. Of note, however, the potential effect of immunomodulation on risk of AS has not yet been studied.The MR approach has recently been extended to estimate the effects of immunomodulation on important clinical outcomes. C-reactive protein (CRP) is a well-established marker of systemic inflammation and is consequently a useful clinical indicator to evaluate the effectiveness of anti-inflammatory treatments. Several studies have proxied the effects of inhibiting IL6 pathway signalling by identifying single-nucleotide polymorphisms (SNPs) that are both significantly associated with CRP levels and located in or near to the The present study uses the MR approach with genetic proxies of the immunomodulatory drugs tocilizumab, canakinumab and colchicine to investigate the potentially causal role of inflammation in AS pathogenesis and to predict whether administration of these drugs would be likely to reduce the risk of developing AS.19. Tocilizumab was selected based on preliminary evidence suggesting a role of IL6 in AS16. The method for identifying genetic proxies and estimating their anti-inflammatory effect was adapted from previously published approaches27 and is summarised in Fig. 31.This study uses a two-sample MR approach with summary data from published studies. Three major anti-inflammatory drugs were considered in this study: tocilizumab, canakinumab and colchicine. Canakinumab and colchicine were selected based on recent trials demonstrating effectiveness for coronary disease32. This included IL1β for canakinumab, IL6R for tocilizumab and TUBB, TUBB2A, TUBB2B, TUBB3, TUBB4A, TUBB4B, TUBB6, TUBB1, TUBB8 and TUBB8B for colchicine. The gene, promoter and enhancer regions for each of these genes were identified using the GeneCards platform33  levels (mg/L) in a genome-wide association study (GWAS) meta-analysis of UK Biobank and Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) study participants28 at a Bonferroni-corrected p value threshold . SNPs were clumped to maximise sensitivity using a linkage disequilibrium threshold of r2 < 0.1 and 1 Mb distance cut-off using the 1000 genomes European reference panel. Palindromic SNPs were excluded. SNPs used to proxy each anti-inflammatory drug are presented in Supplementary Table Genes encoding the protein targets of the anti-inflammatory drugs under investigation were identified using the DrugBank database28. This is the largest GWAS on CRP to date and includes 575,531 participants of European descent. Further details are available in Supplementary Table Summary statistics for the association between the SNPs identified above and CRP levels were extracted from the GWAS meta-analysis of UK Biobank and CHARGE participants35. A summary of the significant SNPs identified in this meta-analysis are reported in Supplementary Table 35.Aortic stenosis was the primary outcome variable. Summary statistics were obtained from a meta-analysis of 10 European cohorts, including 13,765 cases and 640,102 controlsThe primary MR analysis was an inverse variance weighted (IVW) random effect model, with results expressed as odds ratios (OR) on the outcome per unit decrease in genetically predicted natural log-transformed CRP level. For exposures with only a single suitable SNP identified, Wald ratios were calculated for the primary analysis.37. Each method makes different assumptions and thus concordance between methods provides confidence in the conclusion. The weighted-median approach assumes that at least half the instrumental variables are valid. Weighted-mode assumes that the most common causal effect is consistent with the true effect. MR-PRESSO consists of three steps: (1) the MR-PRESSO global test evaluates for horizontal pleiotropy; (2) calculation of the outlier-corrected causal estimate which corrects for horizonal pleiotropy that has been detected; and (3) the MR-PRESSO distortion test, which evaluates whether the causal estimate differs significantly after correction for outliers at p < 0.05 threshold37. MR-Egger uses the Instrument Strength Independent of Direct Effect assumption, which states that pleiotropic effects from the genetic variants to the outcome are independent of the association between genetic variants and exposure. Under this assumption, the intercept from MR-Egger analysis estimates the average pleiotropic effect of the genetic variants38. MR-RAPS is robust to idiosyncratic and systematic pleiotropy36. Leave-one-out sensitivity analysis involves removing a single variant from the analysis in turn then assessing the fluctuation in the estimate for possible outlier contribution. Outliers were further identified using Cook’s distance method and the above analyses re-performed using an outlier-excluded dataset. Heterogeneity of the genetic variants was assessed using scatter plots, funnel plots and applying Cochran’s Q-statistic.To evaluate the robustness of findings, we performed several sensitivity analyses including weighted-median, weighted-mode, MR-PRESSO, MR-Egger, MR-Robust Adjusted Profile Score (MR-RAPS) and leave-one-out sensitivity analyses29. Osteoarthritis (OA) was used as the negative control outcome, with summary statistics obtained from a GWAS of European ancestry UK Biobank participants that included 10,083 cases and 40,425 controls30. Additionally, analyses were conducted for coronary artery disease (CAD) as the outcome to compare with the results for AS. These analyses used summary statistics from a large GWAS meta-analysis of 48 studies including 60,801 coronary artery disease cases and 123,504 controls39.Positive and negative controls were included. Rheumatoid arthritis (RA) was used as the positive control outcome, with summary statistics obtained from a large GWAS meta-analysis that included 22,686 RA cases and 288,644 controls 40.Statistical analyses were performed using R version 1.4.1106There were 18, 9 and 1 suitable SNPs identified to proxy the effects of tocilizumab (IL6R antagonist), canakinumab (IL1β inhibitor) and colchicine (β-tubulin inhibitor), respectively  Fig. . This waGenetically proxied inhibition of IL1β did not meet statistical significance for association with reduced risk of AS on IVW primary analysis  Fig.  and therGenetically proxied inhibition of β-tubulin was associated with increased risk of AS by Wald ratio analysis . This was based on identification of a single suitable SNP and consequently sensitivity analyses could not be performed.There was a significant association between genetically predicted lower CRP and AS on IVW primary analysis  Fig.  and 4. HThe positive and negative controls corresponded to broadly expected results. Significant associations were identified between genetically proxied IL6R and IL1β mediated lower CRP levels and reduced risk of RA on primary IVW  and sensitivity analyses. There was no association identified between genetically proxied β-tubulin inhibition and RA on Wald ratio . There was a significant association between genetically proxied reduced CRP and risk of RA on primary IVW analysis  but this was not robust to sensitivity analyses  and sensitivity analyses. There was no robust association between genetically proxied IL1β, β-tubulin or overall CRP and risk of CAD  in 10,061 patients with previous myocardial infarction and elevated CRP demonstrated that IL1β inhibition with canakinumab was associated with reduced CRP levels and an associated reduction in cardiovascular events, independent of lipids and blood pressure41. Concordantly, the Cardiovascular Inflammation Reduction Trial in 4786 patients with previous myocardial infarction or multivessel coronary disease showed no reduction in IL1β, IL6 or CRP levels and correspondingly did not show any reduction in cardiovascular events among patients treated with methotrexate compared to placebo42. The Colchicine Cardiovascular Outcomes Trial (COLCOT) of 4745 patients with recent myocardial infarction demonstrated lower rates of further cardiovascular events in patients treated with colchicine compared to placebo, and the Low-Dose Colchicine trial in 5522 patients with chronic coronary disease similarly demonstrated reduced cardiovascular event rates in patients treated with colchicine compared to placebo19. While the present study did find a statistically significant association between genetically predicted colchicine and AS on Wald ratio analysis, the identification of only a single suitable SNP for analysis means that sensitivity analyses could not be performed. Notably, the identified SNP rs56283750 was identified as being in the promoter/enhancer region for the TUBB3 gene, which is typically expressed in neuronal and testicular tissue, rather than cardiac tissue43. Moreover there are several isoforms within the β-tubulin family, and therefore genetic downregulation of one isoform may be compensated by upregulation of the others. If such compensation occurs, then a SNP in a given isoform would be unlikely to result in significantly altered peripheral CRP levels or clinically significant diseases. Additionally, the effects of colchicine are pleiotropic, incompletely understood and may not be adequately captured by reduction in CRP44. Further work is thus required to investigate the potential role for colchicine in AS.AS is known to share many risk factors with atherosclerosis. Current American and European guidelines recommend treatment of coexistent hypertension, and American guidelines additionally recommend statin therapy in patients with calcific AS and renin-angiotensin system blockade in patients who have undergone a transcatheter aortic valve implantation (TAVI)45. In the larger Framingham Heart Study’s offspring cohort of 2683 participants, the group with aortic and/or mitral calcification had elevated levels of CRP and IL6, although this was no longer significant after adjustment for traditional cardiovascular risk factors46. In a histopathological study of 103 human aortic valves explanted at the time of aortic valve replacement, patients taking angiotensin receptor blockers had significantly lower fibrosis scores and IL6 expression but no difference in the amount of valve calcification16. In addition to blood pressure reduction, this anti-inflammatory and anti-fibrotic effect may be a contributing explanation to the previous observational findings that treatment with renin-angiotensin system blockade is associated with reduced mortality after TAVI48. A further histopathological investigation of 46 stenotic aortic valves explanted at the time of aortic valve replacement compared to 10 non-calcified controls with normal echocardiograms at the time of heart transplantation found that IL6 levels were nine times higher in the calcified compared to control valves15. A mouse model of AS induced by mechanical wire injury similarly demonstrated marked elevations in the inflammatory cytokines IL1β, IL6 and tumour necrosis factor α49. Consistent with this, an investigation of mice deficient in IL1 receptor antagonist demonstrated increased aortic valve leaflet thickness and transvalvular velocities compared to wild-type mice. This finding was independent of systolic blood pressure and lipids50.There are no randomised trials of immunomodulatory medications in AS yet. Several observational studies have demonstrated an association between elevated inflammatory markers and AS. In a small study of 141 patients, elevated CRP was associated with AS, even after adjustment for traditional cardiovascular risk factors including hypertension and dyslipidemia52. This has important clinical implications, since treatments which target earlier stages may not be effective for late-stage disease and vice versa. IL6 is downstream of IL1β in the NF-kB pathway and upregulation of this pathway is thought to promote the osteogenic transition of interstitial cells that leads to valve mineralisation52. Our results suggest that immunomodulatory treatments that reduce IL6R signalling may therefore be expected to hinder AS at this stage in the pathogenesis. The neutral result from the IL1β analysis in the present study is notable given that IL1β is upstream of IL6R, and previously reported evidence supports a protective effect of canakinumab for patients with coronary disease. There are several possible explanations for this, including the smaller number of SNPs available for analysis for IL1β compared to IL6R. Furthermore, currently available data does not facilitate targeted MR analyses to distinguish factors which contribute to disease incidence versus progression, which would have important therapeutic implications for AS. This might be evaluated in future by GWAS analyses that are powered to evaluate cohorts with different AS severities. Additionally, the administration of immunomodulatory drugs is not without risk. The CANTOS trial, for example, demonstrated that canakinumab is associated with higher incidence of fatal infection compared to placebo17. Of note, this did not translate to a significant difference in all-cause mortality, and canakinumab importantly reduced the primary endpoint composite of nonfatal myocardial infarction, nonfatal stroke and cardiovascular death. Nevertheless, this does highlight the importance of considering the potential risks of immunomodulation along with the intended benefit.The current conception of AS pathogenesis involves endothelial injury and dysfunction, lipid deposition, immune cell infiltration, osteogenic transition of valve interstitial cells and valve mineralisation46. Similarly lipoprotein(a), which is linked with pro-inflammatory effects, has been associated with increased risk of AS, and notably the relationship between lipoprotein(a) and AS is only modestly modulated by CRP levels53. Furthermore there is genetic evidence supporting a potentially causal relationship between lipoprotein(a) and AS35, yet MR studies evaluating CRP in diseases such as coronary artery disease have repeatedly shown no evidence of a causal role55. This is likely because CRP is a downstream inflammatory molecule. Hence CRP is expected to be elevated when upstream inflammatory processes are active, but CRP does not necessarily have a causal role in the pathogenic inflammation of coronary disease or AS itself. This would explain the observed elevation of CRP in AS and coronary disease, while also explaining the repeatedly negative results of MR analyses which aim to evaluate for a causal relationship between elevated CRP and coronary disease54. This is also the reason why it is reasonable to use CRP to identify relevant proxies for tocilizumab and canakinumab, as performed in the present study, since SNPs which reduce the function of IL6R and IL1β can be identified as those which reduce the levels of downstream molecules such as CRP. While an alternative methodological approach to the present study might have been to use GWAS analyses of peripheral IL6R and IL1β protein levels instead of CRP, there is currently a paucity of well-powered GWAS studies that analyse these proteins, with the available studies being at least an order of magnitude smaller than the CRP GWAS used in the present analysis. Moreover, the approach of using CRP as a downstream marker to identify genetic proxies has been identified as a suitable methodology in several previous studies25. Notably, however, whereas the analyses for IL6R and IL1β used CRP as a downstream marker to identify relevant genetic proxies, the analysis of overall CRP in the present study evaluated whether CRP itself has a causal role in AS. While the positive results from the primary IVW analysis suggest that CRP might have a causal role, there was notably significant heterogeneity identified in the IVW analysis, pleiotropy identified by the Egger intercept and the results of the weighted median, weighted mode and MR-Egger analyses were discordant with the primary IVW analysis. Taken together with previously published literature, it is less convincing that CRP itself has a pathogenic role in AS. Rather, the main utility of CRP appears to remain as a marker of active upstream inflammatory processes.The finding in the current study that overall CRP is associated with reduced risk of AS on IVW analysis should be interpreted cautiously given that the sensitivity analyses did not support a significant association, and there was evidence of significant heterogeneity and pleiotropy. Previous observational clinical studies have demonstrated associations between elevated CRP and ASThe present study investigates the potential effects of immunomodulation through drug proxies on AS. A strength of this analysis is that the MR results estimate the cumulative lifelong effect of genetically reduced inflammation. This is particularly relevant since inflammation is thought to occur early in the development of AS. Furthermore, since AS is a chronic condition with a long subclinical stage, a traditional randomised controlled trial will be costly and logistically challenging given the long follow-up time that will be required. However, it is unclear whether the results of the present study can be generalised to patients with established AS. It is possible, for example, that immunomodulation may be more effective, or even exclusively effective, at the initiation of AS pathogenesis.The present study suggests that inflammation may have a causal role in the development of AS. Through genetic drug proxies, we demonstrate that inhibition of IL6R may reduce the risk of AS. In the absence of randomised trials, this study provides the most compelling evidence to date for the potential role of immunomodulation in the prevention of AS.Supplementary Figures.Supplementary Tables.