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Grant, S. F. A.

Publications and source records attributed to Grant, S. F. A..

13 recordsLinked to original sources

Identifying Putative Pathogenic Non-Coding Variants in Unresolved Rare Disease Patients Using Topologically Associated Domains

Unresolved rare disease is a major public health challenge affecting ~300 million people worldwide. At least 50% of these individuals remain genetically unresolved after applying exome sequencing and/or whole genome sequencing. One source of these missing diagnoses is the presence of rare variants within the non-coding genome that are detected but not interpreted by whole genome sequencing. In order to systematically evaluate candidate pathogenic non-coding variants, we created the Genomic Analysis of Variants in Unresolved Rare Disease (GAVURD) system. GAVURD leverages trio whole genome sequencing alignment data to produce a short list of putative pathogenic non-coding variants for a given proband. GAVURD uses best practices for de novo and rare inherited variant identification, links variants to human disease genes harnessing topologically associated domain (TAD) data, and rank prioritizes variants based on phenotypic overlap. As a proof-of-concept, we applied GAVURD to ten probands with unresolved rare disease and implicated six potentially causal non-coding variants based on a confluence of evidence supportive of pathogenicity. The GAVURD system serves an important role in prioritizing candidate non-coding causal variants for unresolved rare disease that can serve as the high value and informed focus of additional functional follow-up studies.

genomics↗

A meta-analysis of chromatin-associated loci provides insights into mechanistic interpretations of trait heritability

The vast majority of trait-associated loci discovered through genome-wide association studies (GWAS) are non-coding, yet most lack statistical alignment with any discovered expression quantitative trait loci (eQTLs). In particular, eQTLs are depleted at gene-distal regions and at "functionally important" genes - those with strong selective constraint and complex regulatory landscapes - likely due to selective depletion of high-effect variants. Here, we investigate the role of variants with weaker effects on expression transmitted through distal regulatory elements, which are detectable as chromatin accessibility QTLs (caQTLs). We aggregated caQTL data from ten studies derived across different tissues, cell-types and lines, representing 104,024 lead caQTLs across 3,457 samples. We found that, across a range of gene properties, caQTLs are discovered at functionally important genes more often than eQTLs. These observations are consistent with a model in which many eQTLs and GWAS hits are mediated through genetic effects on regulatory elements, which may have weak or context-dependent effects on gene expression. Our results suggest that caQTL discovery is more sensitive than eQTL discovery in capturing the molecular consequences of GWAS hits, and can provide complimentary information to eQTLs by implicating functional mechanisms of additional disease-associated loci.

genetics↗

Variant-to-gene mapping identifies ARHGEF12 as a primary open-angle glaucoma effector gene

Primary open-angle glaucoma (POAG), a leading cause of irreversible blindness worldwide, has a strong genetic basis. The Primary Open-Angle African Ancestry Glaucoma Genetics study previously identified 46 risk loci associated with this disease. To pinpoint causal variants and their corresponding effector genes, we analyzed gene expression, chromatin accessibility, and conformation in two ocular cell types: trabecular meshwork cells (hTMCs) and retinal ganglion cells derived from induced pluripotent stem cells (hiPSC-RGCs). We identified 24 candidate genes in hTMCs and 56 in hiPSC-RGCs. Several prioritized genes showed altered mRNA expression, implicating pathways involved in oxidative stress, tissue homeostasis, neuronal maintenance, and retinal degeneration. Among these genes, ARHGEF12 was selected for further validation because local and distal promoter interactions nominated it in both cell types, and prior evidence reproducibly links it to POAG. While its role in hTMCs is established, its function in RGCs is unclear. hiPSC-RGCs generated from a POAG donor homozygous for the risk allele exhibited reduced ARHGEF12 expression, altered morphology, and disrupted neuronal activity. By linking POAG risk loci to candidate effector genes in ocular cell types, this study provides a framework for functional evaluation of additional POAG risk variants.

genomics↗

Cross-species variant-to-function analyses implicate MEIS1 in conferring sleep abnormalities and impaired cerebellar development

Genome wide association study (GWAS) reports substantially outpace subsequent functional characterization. Pinpointing the causal effector gene(s) at GWAS loci remains challenging given the non-coding genomic residency of >98% of these signals. We previously implicated effector genes at GWAS loci for the complex and polygenic disorder of human insomnia using a high-resolution cell-specific, chromatin capture-based variant-to-gene mapping protocol, paired with sleep phenotyping in Drosophila. In this study, we leveraged a diurnal vertebrate model with higher genomic conservation, namely zebrafish, to screen our six highest confidence candidate genes and identify those whose loss-of-function impaired sleep characteristics related to human insomnia-like behaviors. Of these genes, we observed that CRISPR-mediated deletion of the zebrafish ortholog of MEIS1 produced nighttime specific sleep fragmentation and increased latency to sleep, pointing to a conserved role for MEIS1 in sleep maintenance. Comparing our human cell-based chromatin accessibility and contact maps with publicly available zebrafish spatial genomic data revealed highly conserved genomic architecture harboring the insomnia GWAS variant of interest. Notably, this genomic conservation was selective for the zebrafish ortholog which contributed to the sleep phenotype, meis1b, while the duplicated ohnolog meis1a proved dispensable. Motivated by this, we characterized the spatio-temporal expression of meis1b in zebrafish, showing it is comparable to human with respect to cerebellar granule progenitors. Ultimately, we found that loss of meis1b impairs cerebellar development. Together, our work provides a powerful model for screening human disorder risk genes for sleep fragmentation using a tractable vertebrate and supports a conserved cerebellar role for MEIS1 in sleep disturbance.

neuroscience↗

Zebrafish screen of high-confidence targets at insomnia GWAS loci implicates MEIS1, SKIV2L, and ARFGAP2 as conserved regulators of sleep-wake behaviors

Recent large-scale human genome-wide association studies (GWAS) for insomnia have identified more than 200 significant loci. The functional relevance of these loci to the pathogenesis of insomnia is largely unknown. GWAS signals are typically non-coding variants, which are often arbitrarily annotated to the nearest protein-coding gene; however, due to 3D chromatin structure, variants can interact with more distal genes driving their function. The distal gene may, therefore, represent the true causal gene influencing the phenotype. By integrating our high-resolution chromatin interaction maps from neural progenitor cells with phenotypic data from a Drosophila RNAi screen, we prioritized candidate genes that we hypothesized would have deep phylogenetic conservation of sleep function. To determine the conservation of these candidate genes in the context of vertebrate sleep and their relevance to insomnia-like behaviors, we performed CRISPR-Cas9 mutagenesis in larval zebrafish for six highly conserved candidate genes and examined sleep-wake behaviors using automated video-tracking. CRISPR mutation of zebrafish orthologs of MEIS1 and SKIV2L produced insomnia-like behaviors, while mutation of ARFGAP2 impaired activity and development in our larval zebrafish model, demonstrating the importance of performing functional validation of GWAS-implicated effector genes to reveal genes influencing disease-relevant mechanisms.

genetics↗

The three-dimensional chromatin structure of the major human pancreatic cell types reveals lineage-specific regulatory architecture of T2D risk

Three-dimensional (3D) chromatin organization maps help to dissect cell type-specific gene regulatory programs. Furthermore, 3D chromatin maps have contributed to elucidating the pathogenesis of complex genetic diseases by connecting distal regulatory regions and genetic risk variants to their respective target genes. To understand the cell type-specific regulatory architecture of diabetes risk, we generated transcriptomic and 3D epigenomic profiles of human pancreatic acinar, alpha, and beta cells using single-cell RNA-seq, single-cell ATAC-seq, and high-resolution Hi-C of sorted cells. Comparisons of these profiles revealed differential A/B (open/closed) chromatin compartmentalization, chromatin looping, and transcriptional factor mediated control of cell type-specific gene regulatory programs. We identified a total of 4,750 putative causal-variant-target-gene pairs at 194 type 2 diabetes GWAS signals using pancreatic 3D chromatin maps. We found that the connections between candidate causal variants and their putative target effector genes are cell-type stratified and emphasize previously underappreciated roles for alpha and acinar cells in diabetes pathogenesis.

genetics↗

Projecting genetic associations through gene expression patterns highlights disease etiology and drug mechanisms

Genes act in concert with each other in specific contexts to perform their functions. Determining how these genes influence complex traits requires a mechanistic understanding of expression regulation across different conditions. It has been shown that this insight is critical for developing new therapies. In this regard, the role of individual genes in disease-relevant mechanisms can be hypothesized with transcriptome-wide association studies (TWAS), which have represented a significant step forward in testing the mediating role of gene expression in GWAS associations. However, modern models of the architecture of complex traits predict that gene-gene interactions play a crucial role in disease origin and progression. Here we introduce PhenoPLIER, a computational approach that maps gene-trait associations and pharmacological perturbation data into a common latent representation for a joint analysis. This representation is based on modules of genes with similar expression patterns across the same conditions. We observed that diseases were significantly associated with gene modules expressed in relevant cell types, and our approach was accurate in predicting known drug-disease pairs and inferring mechanisms of action. Furthermore, using a CRISPR screen to analyze lipid regulation, we found that functionally important players lacked TWAS associations but were prioritized in trait-associated modules by PhenoPLIER. By incorporating groups of co-expressed genes, PhenoPLIER can contextualize genetic associations and reveal potential targets missed by single-gene strategies.

bioinformatics↗

A UVB-responsive common variant at chr7p21.1 confers tanning response and melanoma risk via regulation of the aryl hydrocarbon receptor gene (AHR)

Genome-wide association studies have identified a melanoma-associated locus on chromosome band 7p21.1 with rs117132860 as the lead SNP, and a secondary independent signal marked by rs73069846. rs117132860 is also associated with tanning ability and cutaneous squamous cell carcinoma (cSCC). As ultraviolet radiation (UVR) is a key environmental exposure for all three traits, we investigated the mechanisms by which this locus contributes to melanoma risk, focusing on cellular response to UVR. Fine-mapping of melanoma GWAS identified four independent sets of candidate causal variants. A GWAS region-focused Capture-C study of primary melanocytes identified physical interactions between two causal sets and the promoter of the aryl hydrocarbon receptor gene (AHR). Subsequent chromatin state annotation, eQTL, and luciferase assays identified rs117132860 as a functional variant and reinforced AHR as a likely causal gene. As AHR plays critical roles in cellular response to dioxin and UVR, we explored links between this SNP and AHR expression after both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and ultraviolet B (UVB) exposure. Allele-specific AHR binding to rs117132860-G was enhanced following both, consistent with predicted weakened AHR binding to the risk/poor-tanning rs117132860-A allele, and allele-preferential AHR expression driven from the protective rs117132860-G allele was observed following UVB exposure. Small deletions surrounding rs117132860 via CRISPR abrogates AHR binding, reduces melanocyte cell growth, and prolongs growth arrest following UVB exposure. These data suggest AHR is a melanoma susceptibility gene at the 7p21.1 risk locus, and rs117132860 is a functional variant within a UVB-responsive element, leading to allelic AHR expression, and altering melanocyte growth phenotypes upon exposure.

genetics↗

Restriction enzyme selection dictates detection range sensitivity in chromatin conformation capture-based variant-to-gene mapping approaches

Promoter-focused chromatin conformation techniques directly detect interactions between gene promoters and distal genomic sequences, providing structural information relevant to gene regulation without the excessive non-genic architectural data generated by full scale Hi-C. 3D promoter interactome maps are crucial for understanding how epigenomic features like histone modifications and open chromatin, or genetic variants identified in genome wide association studies (GWAS), contribute to biological function. However, variation in sensitivity between such promoter-focused methods, principally due to restriction enzyme selection, has not been systematically assessed. Here, we performed a head-to-head comparison between promoter capture C (PCC) and promoter capture Hi-C (PCHiC) with the respective 4 cutters DpnII and MboI versus 6 cutter HindIII datasets from the same five cell types. While PCHiC generally produces a higher signal to noise ratio for significant interactions in comparison to PCC, we show that DpnII/MboI detects more proximal interactions and shows little overlap with the HindIII detection range. Promoter-interacting genomic regions mapped by 4-cutters are more enriched for regulatory features and disease-associated genetic variation than 6-cutters maps, suggesting that high-resolution maps better capture gene regulatory architectures than do lower resolution approaches.

genomics↗

Integrative genomics identifies lncRNA regulatory networks across 1,044 pediatric leukemias and solid tumors

Long non-coding RNAs (lncRNAs) play an important role in gene regulation and contribute to tumorigenesis. While pan-cancer studies of lncRNA expression have been performed for adult malignancies, the lncRNA landscape across pediatric cancers remains largely uncharted. Here, we curate RNA sequencing data for 1,044 pediatric leukemia and solid tumors and integrate paired tumor whole genome sequencing and epigenetic data in relevant cell line models to explore lncRNA expression, regulation, and association with cancer. We report a total of 2,657 robustly expressed lncRNAs across six pediatric cancers, including 1,142 exhibiting histotype-specific expression. DNA copy number alterations contributed to lncRNA dysregulation at a proportion comparable to protein coding genes. Application of a multi-dimensional framework to identify and prioritize lncRNAs impacting gene networks revealed that lncRNAs dysregulated in pediatric cancer are associated with proliferation, metabolism, and DNA damage hallmarks. Analysis of upstream regulation via cell-type specific transcription factors further implicated distinct histotype-specific and developmental lncRNAs. We integrated our analyses to prioritize lncRNAs for experimental validation and showed that silencing of TBX2-AS1, our top-prioritized neuroblastoma-specific lncRNA, resulted in significant growth inhibition of neuroblastoma cells, confirming our computational predictions. Taken together, these data provide a comprehensive characterization of lncRNA regulation and function in pediatric cancers and pave the way for future mechanistic studies.

cancer biology↗

Cis-regulatory architecture of human ESC-derived hypothalamic neuron differentiation aids in variant-to-gene mapping of relevant common complex traits

SummaryThe hypothalamus regulates metabolic homeostasis by influencing behavior, energy utilization and endocrine systems. Given its role governing health-relevant traits, such as body weight and reproductive timing, understanding the genetic regulation of hypothalamic development and function should yield insights into these traits and diseases. However, given its inaccessibility, studying human hypothalamic gene regulation has proven challenging. To address this gap, we generated a chromatin architecture atlas of an established embryonic stem cell (ESC)-derived hypothalamic-like neuron (HN) model across three stages of in vitro differentiation. We profiled accessible chromatin and identified physically interacting contacts between gene promoters and their putative cis-regulatory elements (cREs) to characterize changes in the gene regulatory landscape during hypothalamic differentiation. Next, we integrated these data with GWAS loci for multiple traits and diseases enriched for heritability in these cells, identifying candidate effector genes and cREs impacting transcription factor binding. Our results reveal common target genes for these traits, potentially identifying core hypothalamic developmental pathways. Our atlas will enable future efforts to determine precise mechanisms underlying hypothalamic development with respect to specific disease pathogenesis.Competing Interest StatementThe authors have declared no competing interest.View Full Text

genomics↗

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↗

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↗