Biology subjects

Hankenson, K. D.

Publications and source records attributed to Hankenson, K. D..

16 recordsLinked to original sources

bioRxiv2026

Thrombospondin-2 deficiency primes the synovial joint for aberrant tissue remodeling and injury response

ObjectiveThis study investigates joint injury-induced angiogenesis and the effects of genetic deficiency of thrombospondin-2 (TSP2), an anti-angiogenic factor, in joint homeostasis and post-traumatic osteoarthritis (PTOA). MethodWe utilized a murine non-invasive anterior cruciate ligament rupture (ACLR) model of PTOA and mined published synovial transcriptomics datasets to investigate injury-induced synovial angiogenesis. Spatial transcriptomics and flow cytometry of TSP2-GFP reporter mice were used to assess injury-induced thrombospondin-2 and its cellular origins in synovium. Global TSP2 knockout mice (TSP2-KO) were used to assess the effect of TSP2 deficiency on early and late stages of PTOA development via molecular imaging of inflammation and angiogenesis, histopathology, micro-computed tomography, Raman spectroscopy, and synovium bulk RNA-sequencing. ResultsIntra-articular angiogenesis peaked at 7d post-ACLR and declined but remained elevated above baseline at 28d post-ACLR. We identified synovial crosstalk between endothelial cells and sublining fibroblasts as a key driver of angiogenesis and source of thrombospondin-2 signaling, with TSP2 primarily upregulated in sublining fibroblasts. TSP2-KO mice exhibited increased peri-articular inflammation at 7d post-ACLR and inferior bone quality. Histopathology revealed greater PTOA severity but paradoxically lower synovitis in TSP2-KOs. Additionally, aberrant structural remodeling of the entire knee joint was observed in uninjured and ACLR TSP2-KO limbs. The uninjured TSP2-KO synovial transcriptome demonstrated elevated immune, fibrotic, and angiogenic activation; however, TSP2-KO and WT synovial transcriptomes partially converged upon injury. ConclusionTSP2 is essential for joint homeostasis and trauma response. Global TSP2 deficiency causes premature OA and worsened PTOA, suggesting that therapeutic targeting with TSP2 mimetic could be used to prevent OA.

physiology↗

bioRxiv2026

Wnt/β-catenin signaling regulates fibrotic atrophy of intra-articular adipose tissue in post-traumatic osteoarthritis

Synovial joints like the knee are home to adipose tissue depots whose anatomy and functions are closely intertwined with that of other intra-articular soft tissues such as synovium, underscoring the growing understanding that joints are multi-tissue organs. Traumatic joint injury and the onset of osteoarthritis (OA) dramatically remodel the intra-articular adipose niche, marked by infiltration of fibrotic tissue postulated to underpin OA-associated joint stiffness and pain, yet we know very little about the disease-associated dynamics of joint adipose remodeling nor the mechanisms driving these phenomena. Here, we employed 2D histomorphometry and spatial transcriptomics, alongside 3D osmium tetroxide-enhanced micro-computed tomography to comprehensively define the spatiotemporal, structural, and transcriptional rewiring of joint adipose tissue in a non-invasive mouse model of post-traumatic osteoarthritis (PTOA). These revealed marked loss of intra-articular adiposity accompanied by expansion of fibroblast-rich, collagen-dense tissue with pro-fibrotic hallmarks and Wnt/{beta}-catenin-enriched gene programs. Joint adipose exhibited a distinct transcriptional signature compared to subcutaneous white adipose tissue, pointing to unique, depot-specific functions. Stromal cells isolated from PTOA joints had heightened baseline expression of fibrotic and Wnt pathway genes and exhibited impaired de novo adipogenesis, in contrast to cells derived from healthy joints. In accordance with the destabilized biomechanics of PTOA joints, in vitro modeling demonstrated that prolonged, injurious loading and perturbed Wnt/{beta}-catenin signaling were convergent anti-adipogenic cues that suppressed lipid droplet formation and adipogenic gene induction, while promoting markers of fibrosis in joint-derived stromal cells. Complementary gain-of-function studies using ex vivo joint adipose explants and in vivo joint injections demonstrated that chronic Wnt/{beta}-catenin activation, as seen in OA joints, is sufficient to diminish the intra-articular adipogenic program and shift adipose to a more fibrotic phenotype, independent of joint injury. Collectively, these findings establish a multi-modal framework for quantifying joint adipose atrophy and implicate aberrant Wnt/{beta}-catenin signaling and pathological mechanical loading as key factors impairing de novo adipogenesis and driving fibrotic remodeling of intra-articular adipose tissue in PTOA.

cell biology↗

bioRxiv2025

Spatial transcriptomic profiling of decalcified murine musculoskeletal samples via Xenium Prime 5K

Successful generation of high-quality spatial transcriptomics data from murine musculoskeletal tissues has been impeded by the challenge of preserving RNA integrity through the harsh tissue processing steps required for histological sectioning. In particular, the need to thoroughly fix and decalcify mineralized tissues has proven problematic. We detail a comprehensive sample processing pipeline for three common murine musculoskeletal tissue samples, enabling high-quality transcript detection via imaging-based spatial transcriptomics using the Xenium Prime 5K platform from 10x Genomics. Our protocol outlines methodological details for transcardiac perfusion, fixation, decalcification, paraffin processing, and a sample co-embedding strategy facilitating anatomically consistent and simultaneous sectioning of multiple samples onto the spatial transcriptomics slide. Rigorous quality control demonstrates high-quality tissue-specific outcomes across intact knee joints, tibiae, and lumbar spines from adult mice. Our pipeline enabled 70-91% high-quality transcripts across synovium, meniscus, patellar tendon, articular cartilage, subchondral bone, cortical bone, bone marrow, muscle, fracture callus, and dorsal root ganglion tissues. The average number of detected transcripts varied markedly between tissue types - soft tissues such as synovium, patellar tendon, muscle, bone marrow, and callus exhibited ~200 - 400 transcript per cell; mineralized tissues such as subchondral bone, meniscus, and cortical bone exhibited ~ 13 - 150 transcripts per cell; highly active neuronal tissues such as dorsal root ganglion neurons yielded 750 - 1100 transcripts per cell. Canonical cell markers within each tissue confirmed successful identification and representation of key cell types. Through rigorous sample quality assessment at multiple stages of processing, this protocol yields high-quality RNA transcript detection while preserving critical anatomical context and will serve as a valuable tool enabling spatial transcriptomic profiling of intact musculoskeletal tissue samples. Lay summarySpatial transcriptomics is a powerful scientific tool that characterizes genetic coding material ("transcripts") of specific cell types in their native anatomical context. However, successfully applying this tool to musculoskeletal tissues has been challenging because preserving transcript integrity in these tissues requires additional care. The authors of this publication developed a specialized method for preparing tissue samples from mice that works with the spatial transcriptomics platform, 10x Genomics Xenium Prime 5K. This protocol improves sample quality while preserving high-quality genetic information in various mouse musculoskeletal tissues, making it easier to explore their functions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/693132v1_ufig1.gif" ALT="Figure 1"> View larger version (87K): org.highwire.dtl.DTLVardef@4c4389org.highwire.dtl.DTLVardef@e607bcorg.highwire.dtl.DTLVardef@5f957org.highwire.dtl.DTLVardef@34ce27_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

The role of mitochondrial complex I in the proinflammatory response to polylactide implants

During the foreign body response, immune cells are metabolically rewired after exposure to breakdown products of various biomaterials, including polylactide (PLA) and polyethylene. Particles of polyethylene interact with Toll-like receptor 4 on macrophages, resulting in increased oxygen consumption that forms reactive oxygen species at complex I of the mitochondrial electron transport chain (mETC). However, PLA degradation products bind to monocarboxylate transporters for downstream signaling with elevated oxygen consumption rates, whose functional implication is unclear and remains inferred from cellular responses to polyethylene biomaterials. By chemically probing the function of the mETC, we show that proinflammatory macrophages activated by exposure to amorphous PLA (aPLA) breakdown products rely on mitochondrial respiration for ATP production, independent of oxygen consumption rates. In contrast, macrophages activated by semi-crystalline PLA (cPLA) breakdown products exhibit a metabolic phenotype wherein ATP levels are unaffected by changing oxygen consumption rates. In subcutaneous implants, the incorporation of metformin in aPLA or cPLA to chemically inhibit complex I did not effectively modulate the proinflammatory response to biomaterials, suggesting that PLA degradation products elicit a distinct metabolic program, thus providing an alternative perspective on the role of mitochondrial respiration in the inflammatory response to biomaterials.

bioengineering↗

bioRxiv2024

GWAS-informed data integration and non-coding CRISPRi screen illuminate genetic etiology of bone mineral density

Over 1,100 independent signals have been identified with genome-wide association studies (GWAS) for bone mineral density (BMD), a key risk factor for mortality-increasing fragility fractures; however, the effector gene(s) for most remain unknown. Informed by a variant-to-gene mapping strategy implicating 89 non-coding elements predicted to regulate osteoblast gene expression at BMD GWAS loci, we executed a single-cell CRISPRi screen in human fetal osteoblasts (hFOBs). The BMD relevance of hFOBs was supported by heritability enrichment from stratified LD-score regression involving 98 cell types grouped into 15 tissues. 23 genes showed perturbation in the screen, with four (ARID5B, CC2D1B, EIF4G2, and NCOA3) exhibiting consistent effects upon siRNA knockdown on three measures of osteoblast maturation and mineralization. Lastly, additional heritability enrichments, genetic correlations, and multi-trait fine-mapping revealed unexpectedly that many BMD GWAS signals are pleiotropic and likely mediate their effects via non-bone tissues. Extending our CRISPRi screening approach to these tissues could play a key role in fully elucidating the etiology of BMD.

genomics↗

bioRxiv2024

CD47 is Required for Mesenchymal Progenitor Proliferation and Fracture Repair

CD47 is a ubiquitous and pleiotropic cell-surface receptor. Disrupting CD47 enhances injury repair in various tissues but the role of CD47 has not been studied in bone injuries. In a murine closed-fracture model, CD47-null mice showed decreased callus bone volume, bone mineral content, and tissue mineral content as assessed by microcomputed tomography 10 days post-fracture, and increased fibrous volume as determined by histology. To understand the cellular basis for this phenotype, mesenchymal progenitors (MSC) were harvested from bone marrow. CD47-null MSC showed decreased large fibroblast colony formation (CFU-F), significantly less proliferation, and fewer cells in S-phase, although osteoblast differentiation was unaffected. However, consistent with prior research, CD47-null endothelial cells showed increased proliferation relative to WT cells. Similarly, in a murine ischemic fracture model, CD47-null mice showed reduced fracture callus bone volume and bone mineral content relative to WT. Consistent with our in vitro results, in vivo EdU labeling showed decreased cell proliferation in the callus of CD47-null mice, while staining for CD31 and endomucin demonstrated increased endothelial cell mass. Finally, WT mice administered a CD47 morpholino, which blocks CD47 protein production, showed a callus phenotype similar to that of non-ischemic and ischemic fractures in CD47-null mice, suggesting the phenotype was not due to developmental changes in the knockout mice. Thus, inhibition of CD47 during bone healing reduces both non-ischemic and ischemic fracture healing, in part, by decreasing MSC proliferation. Furthermore, the increase in endothelial cell proliferation and early blood vessel density caused by CD47 disruption is not sufficient to overcome MSC dysfunction.

molecular biology↗

bioRxiv2024

Gustatory-neuron-supplied R-spondin-2 is required for taste bud replenishment

Taste buds undergo continuous cell turnover throughout life, and taste cell replenishment relies strictly on innervation, a phenomenon first described almost 150 years ago. Recently, we provided evidence that R-spondin 2 (Rspo2) may be the long-sought gustatory neuron-supplied factor that regulates taste stem cell activity, via its interaction with taste stem/progenitor cell-expressed receptor Rnf43/Znrf3. Yet, whether gustatory-neuron-supplied Rspo2 is strictly required for taste tissue maintenance has not been resolved. Here, we set out to determine the necessity of gustatory-neuron-supplied Rspo2 in taste tissue homeostasis using genetic approaches. We used a mouse line that harbors the neomycin-resistance gene (NeoR) in one of the intron regions of the Rspo2 gene, which results in reduced expression of Rspo2. The number of taste buds is significantly reduced in these mice, compared to wild-type mice, in both anterior and posterior tongue. This phenotypic change was completely reversed by removing NeoR from the Rspo2 gene, thus making it normal. We also combined adeno-associated virus (AAV)-based delivery of Cre recombinase with a mouse line amenable to Cre-based ablation of the Rspo2 exons encoding the receptor-binding domains. Such deletion of Rspo2 in the nodose-petrosal-jugular ganglion complex led to nearly complete loss of taste buds in the circumvallate papilla. Thus, we demonstrate that Rspo2 is the long-sought gustatory-neuron-supplied factor that acts on taste stem cells to maintain taste tissue homeostasis. SignificanceWe have known for 150 years that innervation is required to induce and maintain cell replacement in taste buds. Until recently, the identity of the inducing factor produced by neurons was unknown. We have shown that R-spondin alone is sufficient to substitute for neuronal input to induce taste bud regeneration. Using a genetic loss-of-function approach, we now demonstrate that gustatory-neuron-expressed Rspo2 is required to maintain taste tissue homeostasis. Altogether, our work reveals that Rspo2 is the long-sought neuron-supplied factor that regulates the activity of taste stem/progenitor cells.

neuroscience↗

bioRxiv2023

A Murine Delayed-Healing Model Associates Immune Response with Functional Bone Regeneration after Trauma

Delayed and non-healing fractures, affecting 5-10% of cases, are associated with prolonged disability and diminished quality of life. Although acute inflammation is required to initiate repair, persistent mechanical instability can sustain maladaptive immune and fibrotic responses that impede regeneration. Existing animal models do not adequately recapitulate mechanical instability, the principal driver of hypertrophic nonunion in clinical settings, thereby limiting translational relevance. In this study, we developed a murine model of delayed fracture healing using tunable intramedullary fixation to impose controlled interfragmentary strain. High-strain conditions (low-stiffness nail, 15-30% strain) produced enlarged calluses characterized by delayed ossification, increased fibrotic tissue (2.9-fold, p = 0.0099), and reduced biomechanical integrity (1.6-fold decrease in stiffness, p = 0.024) relative to low-strain controls (high-stiffness nail, <5% strain). Spatial transcriptomic analysis identified persistent fibrotic niches in high-strain calluses enriched with fibroblast-associated genes (e.g., Pdgfrb, Lgals3) and dysregulated macrophage-fibroblast signaling (Spp1, Mmp9). Systemically, high-strain fractures were associated with distinct immune signatures in which CD206 macrophages and CD25 regulatory T cells predicted healing outcomes (R = 0.72, p = 0.004), suggesting early immune polarization as a determinant of repair trajectory. Elevated CD8 T cell responses were also observed, consistent with a sustained inflammatory state associated with impaired healing. These findings identify mechanical instability as a driver of pathological immune-stromal interactions and establish a preclinical platform for investigating mechanobiology-informed therapeutic strategies. This work supports a conceptual framework in which hypertrophic nonunion is understood as a disorder arising from dysregulated interactions between mechanical cues and immune responses. TeaserMechanical strain hijacks immune signaling to induce fibrosis and block bone regeneration in unstable fractures.

bioengineering↗

bioRxiv2023

Regulating the proinflammatory response to implanted composite biomaterials comprising polylactide and hydroxyapatite by targeting immunometabolism

Composite biomaterials comprising polylactide (PLA) and hydroxyapatite (HA) are applied in bone, cartilage and dental regenerative medicine, where HA confers osteoconductive properties. However, after surgical implantation, adverse immune responses to these composites can occur, which have been attributed to size and morphology of HA particles. Approaches to effectively modulate these adverse immune responses have not been described. PLA degradation products have been shown to alter immune cell metabolism, which drives the inflammatory response. Therefore, we aimed to modulate the inflammatory response to composite biomaterials by regulating glycolytic flux with small molecule inhibitors incorporated into composites comprised of amorphous PLA (aPLA) and HA (aPLA+HA). Inhibition at specific steps in glycolysis reduced proinflammatory (CD86+CD206-) and increased pro-regenerative (CD206+) immune cell populations around implanted aPLA+HA resulting in a pro-regenerative microenvironment. Notably, neutrophil and dendritic cell (DC) numbers along with proinflammatory monocyte and macrophage populations were decreased, and Arginase 1 expression among DCs was increased. Targeting immunometabolism to control the inflammatory response to biomaterial composites, and creating a pro-regenerative microenvironment, is a significant advance in tissue engineering where immunomodulation enhances osseointegration, and angiogenesis, which will lead to improved bone regeneration.

bioengineering↗

bioRxiv2022

Wnt-associated adult stem cell marker Lgr6 is required for osteogenesis and fracture healing

Despite the remarkable regenerative capacity of skeletal tissues, nonunion of bone and failure of fractures to heal properly presents a significant clinical concern. Stem and progenitor cells are present in bone and become activated following injury; thus, elucidating mechanisms that promote adult stem cell-mediated healing is important. Wnt-associated adult stem marker Lgr6 is implicated in the regeneration of tissues with well-defined stem cell niches in stem cell-reliant organs. Here, we demonstrate that Lgr6 is dynamically expressed in osteoprogenitors in response to fracture injury. Using an Lgr6-null mouse model, we find that Lgr6 expression is necessary for maintaining bone volume and efficient postnatal bone regeneration in adult mice. Skeletal progenitors isolated from Lgr6-null mice have reduced colony-forming potential and reduced osteogenic differentiation capacity due to attenuated cWnt signaling. Lgr6-null mice consist of a lower proportion of self-renewing stem cells. In response to fracture injury, Lgr6-null mice have deficient proliferation of periosteal progenitors and reduced ALP activity. Further, analysis of bone regeneration phase and remodeling phase of fracture healing in Lgr6-null mice showed impaired endochondral ossification and reduced mineralization. We propose that in contrast to not being required for successful skeletal development Lgr6-positive cells have a direct role in endochondral bone repair.

cell biology↗

bioRxiv2022

Stereochemistry Determines Immune Cellular Responses to Polylactide Implants

Repeating L- and D-chiral configurations determine polylactide (PLA) stereochemistry which affects its thermal and physicochemical properties, including degradation profiles. Clinically, degradation of implanted PLA biomaterials promotes prolonged inflammation and excessive fibrosis, but the role of PLA stereochemistry is unclear. Additionally, although PLA of varied stereochemistries cause differential immune responses in-vivo, this observation has yet to be effectively modeled in-vitro. A bioenergetic model was applied to study immune cellular responses to PLA containing > 99% L-lactide (PLLA), > 99% D-lactide (PDLA) and a 50/50 melt-blend of PLLA and PDLA (stereocomplex PLA). Stereocomplex PLA breakdown products increased IL-1{beta}, TNF- and IL-6 protein levels but not MCP-1. Expression of these proinflammatory cytokines is mechanistically driven by increases in glycolysis in primary macrophages. In contrast, PLLA and PDLA degradation products selectively increase MCP-1 protein expression. Whereas both oxidative phosphorylation and glycolysis are increased with PDLA, only oxidative phosphorylation is increased with PLLA. For each biomaterial, glycolytic inhibition reduces proinflammatory cytokines and markedly increases anti-inflammatory (IL-10) protein levels; differential metabolic changes in fibroblasts were observed. These findings provide mechanistic explanations for the diverse immune responses to PLA of different stereochemistries, and underscore the pivotal role of immunometabolism on the biocompatibility of biomaterials applied in medicine.

bioengineering↗

bioRxiv2022

Polylactide Degradation Activates Immune Cells by Metabolic Reprogramming

Polylactide (PLA) is the most widely utilized biopolymer in medicine. However, chronic inflammation and excessive fibrosis resulting from its degradation remain significant obstacles to extended clinical use. Immune cell activation has been correlated to the acidity of breakdown products, yet methods to neutralize the pH have not significantly reduced adverse responses. Using a bioenergetic model, we observed delayed cellular changes that were not apparent in the short-term. Amorphous and semi-crystalline PLA degradation products, including monomeric L-lactic acid, mechanistically remodel metabolism in cells leading to a reactive immune microenvironment characterized by elevated proinflammatory cytokines. Selective inhibition of metabolic reprogramming and altered bioenergetics both reduce these undesirable high cytokine levels and stimulate anti-inflammatory signals. Our results present a new biocompatibility paradigm by identifying metabolism as a target for immunomodulation to increase tolerance to biomaterials, ensuring safe clinical application of PLA-based implants for soft- and hard-tissue regeneration, and advancing nanomedicine and drug delivery.

bioengineering↗

bioRxiv2022

Synovial fibroblasts assume distinct functional identities and secrete R-spondin 2 to drive osteoarthritis

ObjectivesSynovium is acutely affected following joint trauma and contributes to post-traumatic osteoarthritis (PTOA) progression. Little is known about discrete cell types and molecular mechanisms in PTOA synovium. We aimed to describe synovial cell populations and their dynamics in PTOA, with a focus on fibroblasts. We also sought to define mechanisms of synovial Wnt/{beta}-catenin signaling, given its emerging importance in arthritis. MethodsWe subjected mice to non-invasive anterior cruciate ligament rupture as a model of human joint injury. We performed single-cell RNA-sequencing to assess synovial cell populations, subjected Wnt-GFP reporter mice to joint injury to study Wnt-active cells, and performed intra-articular injections of the Wnt agonist R-spondin 2 (Rspo2) to assess whether gain-of-function induced pathologies characteristic of PTOA. Lastly, we used cultured fibroblasts, macrophages, and chondrocytes to study how Rspo2 orchestrates crosstalk between joint cell types. ResultsWe uncovered seven distinct functional subsets of synovial fibroblasts in healthy and injured synovium, and defined their temporal dynamics in early and established PTOA. Wnt/{beta}-catenin signaling was overactive in PTOA synovium, and Rspo2 was strongly induced after injury and secreted exclusively by Prg4hi lining fibroblasts. Trajectory analyses predicted that Prg4hi lining fibroblasts arise from a pool of Dpp4+ mesenchymal progenitors in synovium, with SOX5 identified as a potential regulator of this emergence. We also showed that Rspo2 orchestrated pathological crosstalk between synovial fibroblasts, macrophages, and chondrocytes. ConclusionsSynovial fibroblasts assume distinct functional identities during PTOA, and Prg4hi lining fibroblasts secrete the Wnt agonist Rspo2 to drive pathological crosstalk in the joint after injury.

cell biology↗

bioRxiv2022

Lipolysis of bone marrow adipocytes is required to fuel bone and the marrow niche during energy deficits

To investigate roles for bone marrow adipocyte (BMAd) lipolysis in bone homeostasis, we created a BMAd-specific Cre mouse model in which we knocked out adipose triglyceride lipase (ATGL, Pnpla2). BMAd-Pnpla2-/- mice have impaired BMAd lipolysis, and increased size and number of BMAds at baseline. Although energy from BMAd lipid stores is largely dispensable when mice are fed ad libitum, BMAd lipolysis is necessary to maintain myelopoiesis and bone mass under caloric restriction. BMAd-specific Pnpla2 deficiency compounds the effects of caloric restriction on loss of trabecular bone, likely due to impaired osteoblast expression of collagen genes and reduced osteoid synthesis. RNA sequencing analysis of bone marrow adipose tissue reveals that caloric restriction induces dramatic elevations in extracellular matrix organization and skeletal development genes, and energy from BMAd is required for these adaptations. BMAd-derived energy supply is also required for bone regeneration upon injury, and maintenance of bone mass with cold exposure.

cell biology↗

bioRxiv2018

Genome-scale Capture C promoter interaction analysis implicates novel effector genes at GWAS loci for bone mineral density

ASBTRACTOsteoporosis is a devastating disease with an essential genetic component. Genome wide association studies (GWAS) have discovered genetic variants robustly associated with bone mineral density (BMD), however they only report genomic signals and not necessarily the precise localization of culprit effector genes. Therefore, we sought to carry out physical and direct variant to gene mapping in a relevant primary human cell type. We developed SPATIaL-seq (genome-Scale, Promoter-focused Analysis of chromaTIn Looping), a massively parallel, high resolution Capture-C based method to simultaneously characterize the genome-wide interactions of all human promoters. By intersecting our SPATIaL-seq and ATAC-seq data from human mesenchymal progenitor cell -derived osteoblasts, we observed consistent contacts between candidate causal variants and putative target gene promoters in open chromatin for ~30% of the 110 BMD loci investigated. Knockdown of two novel implicated genes, ING3 at CPED1-WNT16 and EPDR1 at STARD3NL, had pronounced inhibitory effects on osteoblastogenesis. Our approach therefore aids target discovery in osteoporosis and can be applied to other common genetic diseases.

genomics↗

bioRxiv2016

Determining causal genes from GWAS signals using topologically associating domains

Genome wide association studies (GWAS) have contributed significantly to the understanding of complex disease genetics. However, GWAS only report associated signals and do not necessarily identify culprit genes. As most signals occur in non-coding regions of the genome, it is often challenging to assign genomic variants to the underlying causal mechanism(s). Topologically associating domains (TADs) are primarily cell-type independent genomic regions that define interactome boundaries and can aid in the designation of limits within which an association most likely impacts gene function. We describe and validate a computational method that uses the genic content of TADs to discover candidate genes. Our method, called \"TAD_Pathways,\" performs a Gene Ontology (GO) analysis over genes that reside within TAD boundaries corresponding to GWAS signals for a given trait or disease. We applied our pipeline to the GWAS catalog entries associated with bone mineral density (BMD), identifying Skeletal System Development (Benjamini-Hochberg adjusted p=1.02x10-5) as the top ranked pathway. In many cases, our method implicated a gene other than the nearest gene. Our molecular experiments describe a novel example: ACP2, implicated at the canonical ARHGAP1 locus. We found ACP2 to be an important regulator of osteoblast metabolism, whereas ARHGAP1 was not supported. Our results via the example of BMD demonstrate how basic principles of three-dimensional genome organization can define biologically informed association windows.

bioinformatics↗