An encyclopedia of enhancer-gene regulatory interactions in the human genome.

Identifying transcriptional enhancers and their target genes is essential for understanding gene regulation and the impact of human genetic variation on disease1-6. Here we create and evaluate a resource of >13 million enhancer-gene regulatory interactions across 352 cell types and tissues, by integrating predictive models, measurements of chromatin state and 3D contacts, and largescale genetic perturbations generated by the ENCODE Consortium7. We first create a systematic benchmarking pipeline to compare predictive models, assembling a dataset of 10,411 elementgene pairs measured in CRISPR perturbation experiments, >30,000 fine-mapped eQTLs, and 569 fine-mapped GWAS variants linked to a likely causal gene. Using this framework, we develop a new predictive model, ENCODE-rE2G, that achieves state-of-the-art performance across multiple prediction tasks, demonstrating a strategy involving iterative perturbations and supervised machine learning to build increasingly accurate predictive models of enhancer regulation. Using the ENCODE-rE2G model, we build an encyclopedia of enhancer-gene regulatory interactions in the human genome, which reveals global properties of enhancer networks, identifies differences in the functions of genes that have more or less complex regulatory landscapes, and improves analyses to link noncoding variants to target genes and cell types for common, complex diseases. By interpreting the model, we find evidence that, beyond enhancer activity and 3D enhancer-promoter contacts, additional features guide enhancerpromoter communication including promoter class and enhancer-enhancer synergy. Altogether, these genome-wide maps of enhancer-gene regulatory interactions, benchmarking software, predictive models, and insights about enhancer function provide a valuable resource for future studies of gene regulation and human genetics.

Leveraging polygenic enrichments of gene features to predict genes underlying complex traits and diseases.

Genome-wide association studies (GWASs) are a valuable tool for understanding the biology of complex human traits and diseases, but associated variants rarely point directly to causal genes. In the present study, we introduce a new method, polygenic priority score (PoPS), that learns trait-relevant gene features, such as cell-type-specific expression, to prioritize genes at GWAS loci. Using a large evaluation set of genes with fine-mapped coding variants, we show that PoPS and the closest gene individually outperform other gene prioritization methods, but observe the best overall performance by combining PoPS with orthogonal methods. Using this combined approach, we prioritize 10,642 unique gene-trait pairs across 113 complex traits and diseases with high precision, finding not only well-established gene-trait relationships but nominating new genes at unresolved loci, such as LGR4 for estimated glomerular filtration rate and CCR7 for deep vein thrombosis. Overall, we demonstrate that PoPS provides a powerful addition to the gene prioritization toolbox.

KLF4 recruits SWI/SNF to increase chromatin accessibility and reprogram the endothelial enhancer landscape under laminar shear stress.

Physiologic laminar shear stress (LSS) induces an endothelial gene expression profile that is vasculo-protective. In this report, we delineate how LSS mediates changes in the epigenetic landscape to promote this beneficial response. We show that under LSS, KLF4 interacts with the SWI/SNF nucleosome remodeling complex to increase accessibility at enhancer sites that promote the expression of homeostatic endothelial genes. By combining molecular and computational approaches we discover enhancers that loop to promoters of KLF4- and LSS-responsive genes that stabilize endothelial cells and suppress inflammation, such as BMPR2, SMAD5, and DUSP5. By linking enhancers to genes that they regulate under physiologic LSS, our work establishes a foundation for interpreting how non-coding DNA variants in these regions might disrupt protective gene expression to influence vascular disease.

SNP-to-gene linking strategies reveal contributions of enhancer-related and candidate master-regulator genes to autoimmune disease.

We assess contributions to autoimmune disease of genes whose regulation is driven by enhancer regions (enhancer-related) and genes that regulate other genes in trans (candidate master-regulator). We link these genes to SNPs using several SNP-to-gene (S2G) strategies and apply heritability analyses to draw three conclusions about 11 autoimmune/blood-related diseases/traits. First, several characterizations of enhancer-related genes using functional genomics data are informative for autoimmune disease heritability after conditioning on a broad set of regulatory annotations. Second, candidate master-regulator genes defined using trans-eQTL in blood are also conditionally informative for autoimmune disease heritability. Third, integrating enhancer-related and master-regulator gene sets with protein-protein interaction (PPI) network information magnified their disease signal. The resulting PPI-enhancer gene score produced >2-fold stronger heritability signal and >2-fold stronger enrichment for drug targets, compared with the recently proposed enhancer domain score. In each case, functionally informed S2G strategies produced 4.1- to 13-fold stronger disease signals than conventional window-based strategies.

Combining SNP-to-gene linking strategies to identify disease genes and assess disease omnigenicity.

Disease-associated single-nucleotide polymorphisms (SNPs) generally do not implicate target genes, as most disease SNPs are regulatory. Many SNP-to-gene (S2G) linking strategies have been developed to link regulatory SNPs to the genes that they regulate in cis. Here, we developed a heritability-based framework for evaluating and combining different S2G strategies to optimize their informativeness for common disease risk. Our optimal combined S2G strategy (cS2G) included seven constituent S2G strategies and achieved a precision of 0.75 and a recall of 0.33, more than doubling the recall of any individual strategy. We applied cS2G to fine-mapping results for 49 UK Biobank diseases/traits to predict 5,095 causal SNP-gene-disease triplets (with S2G-derived functional interpretation) with high confidence. We further applied cS2G to provide an empirical assessment of disease omnigenicity; we determined that the top 1% of genes explained roughly half of the SNP heritability linked to all genes and that gene-level architectures vary with variant allele frequency.

Compatibility rules of human enhancer and promoter sequences.

Gene regulation in the human genome is controlled by distal enhancers that activate specific nearby promoters1. A proposed model for this specificity is that promoters have sequence-encoded preferences for certain enhancers, for example, mediated by interacting sets of transcription factors or cofactors2. This 'biochemical compatibility' model has been supported by observations at individual human promoters and by genome-wide measurements in Drosophila3-9. However, the degree to which human enhancers and promoters are intrinsically compatible has not yet been systematically measured, and how their activities combine to control RNA expression remains unclear. Here we design a high-throughput reporter assay called enhancer × promoter self-transcribing active regulatory region sequencing (ExP STARR-seq) and applied it to examine the combinatorial compatibilities of 1,000 enhancer and 1,000 promoter sequences in human K562 cells. We identify simple rules for enhancer-promoter compatibility, whereby most enhancers activate all promoters by similar amounts, and intrinsic enhancer and promoter activities multiplicatively combine to determine RNA output (R2 = 0.82). In addition, two classes of enhancers and promoters show subtle preferential effects. Promoters of housekeeping genes contain built-in activating motifs for factors such as GABPA and YY1, which decrease the responsiveness of promoters to distal enhancers. Promoters of variably expressed genes lack these motifs and show stronger responsiveness to enhancers. Together, this systematic assessment of enhancer-promoter compatibility suggests a multiplicative model tuned by enhancer and promoter class to control gene transcription in the human genome.

Genome-wide enhancer maps link risk variants to disease genes.

Genome-wide association studies (GWAS) have identified thousands of noncoding loci that are associated with human diseases and complex traits, each of which could reveal insights into the mechanisms of disease1. Many of the underlying causal variants may affect enhancers2,3, but we lack accurate maps of enhancers and their target genes to interpret such variants. We recently developed the activity-by-contact (ABC) model to predict which enhancers regulate which genes and validated the model using CRISPR perturbations in several cell types4. Here we apply this ABC model to create enhancer-gene maps in 131 human cell types and tissues, and use these maps to interpret the functions of GWAS variants. Across 72 diseases and complex traits, ABC links 5,036 GWAS signals to 2,249 unique genes, including a class of 577 genes that appear to influence multiple phenotypes through variants in enhancers that act in different cell types. In inflammatory bowel disease (IBD), causal variants are enriched in predicted enhancers by more than 20-fold in particular cell types such as dendritic cells, and ABC achieves higher precision than other regulatory methods at connecting noncoding variants to target genes. These variant-to-function maps reveal an enhancer that contains an IBD risk variant and that regulates the expression of PPIF to alter the membrane potential of mitochondria in macrophages. Our study reveals principles of genome regulation, identifies genes that affect IBD and provides a resource and generalizable strategy to connect risk variants of common diseases to their molecular and cellular functions.

HyPR-seq: Single-cell quantification of chosen RNAs via hybridization and sequencing of DNA probes.

Single-cell quantification of RNAs is important for understanding cellular heterogeneity and gene regulation, yet current approaches suffer from low sensitivity for individual transcripts, limiting their utility for many applications. Here we present Hybridization of Probes to RNA for sequencing (HyPR-seq), a method to sensitively quantify the expression of hundreds of chosen genes in single cells. HyPR-seq involves hybridizing DNA probes to RNA, distributing cells into nanoliter droplets, amplifying the probes with PCR, and sequencing the amplicons to quantify the expression of chosen genes. HyPR-seq achieves high sensitivity for individual transcripts, detects nonpolyadenylated and low-abundance transcripts, and can profile more than 100,000 single cells. We demonstrate how HyPR-seq can profile the effects of CRISPR perturbations in pooled screens, detect time-resolved changes in gene expression via measurements of gene introns, and detect rare transcripts and quantify cell-type frequencies in tissue using low-abundance marker genes. By directing sequencing power to genes of interest and sensitively quantifying individual transcripts, HyPR-seq reduces costs by up to 100-fold compared to whole-transcriptome single-cell RNA-sequencing, making HyPR-seq a powerful method for targeted RNA profiling in single cells.

Inherited causes of clonal haematopoiesis in 97,691 whole genomes.

Age is the dominant risk factor for most chronic human diseases, but the mechanisms through which ageing confers this risk are largely unknown1. The age-related acquisition of somatic mutations that lead to clonal expansion in regenerating haematopoietic stem cell populations has recently been associated with both haematological cancer2-4 and coronary heart disease5-this phenomenon is termed clonal haematopoiesis of indeterminate potential (CHIP)6. Simultaneous analyses of germline and somatic whole-genome sequences provide the opportunity to identify root causes of CHIP. Here we analyse high-coverage whole-genome sequences from 97,691 participants of diverse ancestries in the National Heart, Lung, and Blood Institute Trans-omics for Precision Medicine (TOPMed) programme, and identify 4,229 individuals with CHIP. We identify associations with blood cell, lipid and inflammatory traits that are specific to different CHIP driver genes. Association of a genome-wide set of germline genetic variants enabled the identification of three genetic loci associated with CHIP status, including one locus at TET2 that was specific to individuals of African ancestry. In silico-informed in vitro evaluation of the TET2 germline locus enabled the identification of a causal variant that disrupts a TET2 distal enhancer, resulting in increased self-renewal of haematopoietic stem cells. Overall, we observe that germline genetic variation shapes haematopoietic stem cell function, leading to CHIP through mechanisms that are specific to clonal haematopoiesis as well as shared mechanisms that lead to somatic mutations across tissues.

Activity-by-contact model of enhancer-promoter regulation from thousands of CRISPR perturbations.

Enhancer elements in the human genome control how genes are expressed in specific cell types and harbor thousands of genetic variants that influence risk for common diseases1-4. Yet, we still do not know how enhancers regulate specific genes, and we lack general rules to predict enhancer-gene connections across cell types5,6. We developed an experimental approach, CRISPRi-FlowFISH, to perturb enhancers in the genome, and we applied it to test >3,500 potential enhancer-gene connections for 30 genes. We found that a simple activity-by-contact model substantially outperformed previous methods at predicting the complex connections in our CRISPR dataset. This activity-by-contact model allows us to construct genome-wide maps of enhancer-gene connections in a given cell type, on the basis of chromatin state measurements. Together, CRISPRi-FlowFISH and the activity-by-contact model provide a systematic approach to map and predict which enhancers regulate which genes, and will help to interpret the functions of the thousands of disease risk variants in the noncoding genome.

Functional disease architectures reveal unique biological role of transposable elements.

Transposable elements (TE) comprise roughly half of the human genome. Though initially derided as junk DNA, they have been widely hypothesized to contribute to the evolution of gene regulation. However, the contribution of TE to the genetic architecture of diseases remains unknown. Here, we analyze data from 41 independent diseases and complex traits to draw three conclusions. First, TE are uniquely informative for disease heritability. Despite overall depletion for heritability (54% of SNPs, 39 ± 2% of heritability), TE explain substantially more heritability than expected based on their depletion for known functional annotations. This implies that TE acquire function in ways that differ from known functional annotations. Second, older TE contribute more to disease heritability, consistent with acquiring biological function. Third, Short Interspersed Nuclear Elements (SINE) are far more enriched for blood traits than for other traits. Our results can help elucidate the biological roles that TE play in the genetic architecture of diseases.

Detecting genome-wide directional effects of transcription factor binding on polygenic disease risk.

Biological interpretation of genome-wide association study data frequently involves assessing whether SNPs linked to a biological process, for example, binding of a transcription factor, show unsigned enrichment for disease signal. However, signed annotations quantifying whether each SNP allele promotes or hinders the biological process can enable stronger statements about disease mechanism. We introduce a method, signed linkage disequilibrium profile regression, for detecting genome-wide directional effects of signed functional annotations on disease risk. We validate the method via simulations and application to molecular quantitative trait loci in blood, recovering known transcriptional regulators. We apply the method to expression quantitative trait loci in 48 Genotype-Tissue Expression tissues, identifying 651 transcription factor-tissue associations including 30 with robust evidence of tissue specificity. We apply the method to 46 diseases and complex traits (average n = 290 K), identifying 77 annotation-trait associations representing 12 independent transcription factor-trait associations, and characterize the underlying transcriptional programs using gene-set enrichment analyses. Our results implicate new causal disease genes and new disease mechanisms.

Diagnostic accuracy and safety of fine-needle aspiration biopsy of the parapharyngeal space.

Fine-needle aspiration (FNA) biopsy of the parapharyngeal space (PPS) is a diagnostic challenge and sampling is often done without image guidance, often trans-orally. Primary PPS tumors are rare, and there is a broad differential diagnosis. The accuracy of PPS FNA, in particular without CT-guidance and using liquid-based cytology (LBC), has not been well studied. Pathology records from our institution (a 1,100 bed Canadian academic tertiary care centre) were searched to identify all patients who underwent PPS FNA from September 1991 to August 2009. The FNA diagnosis was compared to the gold standard of subsequent histopathology or long-term clinical follow-up. Of 36 FNAs, 3 employed image guidance. Eleven (31%) FNAs were nondiagnostic. In the 25 diagnostic FNAs, there was sensitivity 89%, specificity 94%, PPV 89%, NPV 94%, and accuracy 92% for the diagnosis of positive or negative for malignancy. A correct specific diagnosis was made in 9/25 (36%) cases. The nondiagnostic rate was significantly higher (P < 0.025) in FNAs prepared as conventional smears (9/17 = 53%) versus LBC (2/19 = 11%). A specific diagnosis was made significantly more often (P < 0.05) with LBC (8/19 = 43%) versus conventional smear (1/17 = 5.9%). One minor complication from FNA occurred. In conclusion, PPS FNA is safe and accurate for the diagnosis of malignancy. The rate of reporting a specific diagnosis is low. Nondiagnostic FNAs are common. There are more specific diagnoses and fewer nondiagnostic tests with LBC than with conventional smears. Improved specimen quality with LBC is likely a factor.

Batting .500: experience with the fibular free flap in the achondroplastic patient.

OBJECTIVES: To describe the use of the fibular free flap in the achondroplastic patient and to report the potential pitfalls of free flap surgery on this group of patients. DESIGN: Retrospective chart analysis. METHODS: We reviewed our experience with two fibular free flaps in this rare situation. RESULTS: This is the largest series of fibular free flaps reported on the achondroplastic patient. An acute adrenal event resulted in hypothermia and hypotension and ultimately led to the failure of our first reconstructive attempt. Corrective measures were taken during the secondary reconstruction to prevent these systemic issues from coming into play, which ultimately led to a successful free fibular transfer. CONCLUSION: Free fibular transfer is possible for facial reconstruction in the achondroplastic patient, and success can be improved if measures are taken to prevent systemic complications of hypothermia and hypotension in the case of an acute adrenal event.

Do we measure up? Is an objective measuring device necessary for the accurate assessment of oral cavity and oropharyngeal lesions?

OBJECTIVES: The T stage of oral cavity and oropharyngeal squamous cell cancer lesions has a significant impact on patient treatment and overall outcome. Determining the presence and size of palpable lymph nodes also plays an important role in the complex staging of oral cancer. Oral cavity lesions and lymph nodes are often assessed by visual approximation and palpation. The focus of this study was to determine if the introduction of a measurement tool (a ruler) changes the T stage of oral cavity and oropharyngeal lesions and the N stage of lymph nodes. MATERIALS/METHODS: Various pieces of felt that represented oral cavity and oropharyngeal lesions were placed on the tongues of cadaver specimens. Several pieces of felt of different shapes were used to represent each T stage in the oral cavity and oropharyngeal tumour staging system (American Joint Committee on Cancer). Pieces of round clay, of differing sizes, were also placed in the neck of one of the cadavers once a subplatysmal flap was raised. These pieces of clay represented the various node sizes. The study participants were four head and neck surgeons, four senior residents, four junior residents, and five medical students. All subjects were asked to visually inspect the oral cavity and oropharynx of the cadaver and approximate, to the nearest 0.5 cm, the size of the lesion. The subjects were then asked to identify the lymph nodes in the same manner. Once the participants had recorded their answers, they were asked to repeat the process with the aid of a ruler and measure the same lesions and lymph node to the nearest 0.5 cm. RESULTS/CONCLUSIONS: In staging of oral cavity and oropharyngeal cancer, the use of a ruler is necessary to increase the accuracy of tumour staging. There was a statistically significant difference in the estimated size of tumours and nodes when using a ruler. The average absolute error using visual estimation of the tumour size was 5.6 mm. When using the ruler, the error was reduced to 1.7 mm. The node size showed the same trend, with average absolute error on visual estimation being reduced from 7.4 mm to 5.2 mm.

Chondrosarcoma of the head and neck: report of 11 cases and literature review.

OBJECTIVE: Chondrosarcoma is a malignant tumour of mesenchymal origin. Chondrosarcomas most commonly occur on axial structures and are rarely found in the head and neck. A review of these tumours was carried out, focusing on management and outcomes. METHODS: Eleven chondrosarcoma cases of the head and neck were retrospectively identified at a tertiary care teaching centre. RESULTS: There were seven males and four females; ages ranged from 18 to 77 years. Specific sites included the larynx (3), trachea (1), petrous apex (2), skull base (2), cervical spine (1), clivus (1), and cavernous sinus region (1). Eight of the 11 patients had grade I disease (73%), whereas the remaining 3 (27%) had grade II tumours. None had metastatic disease at presentation. Surgical resection with postoperative radiation was the most widely employed primary treatment (55%); the remaining patients (45%) had surgical resection only. There were two recurrences. Salvage surgeries were performed in both. Disease-specific survival was 73% at 5 years. CONCLUSION: Chondrosarcomas are rare tumours of the head and neck. Treatment should be aimed at complete surgical resection with the option of postoperative radiotherapy. They usually portend a favourable long-term prognosis.

Sentinel node biopsy in N0 squamous cell carcinoma of the oral cavity and oropharynx in patients previously treated with surgery or radiation therapy: a pilot study.

OBJECTIVES: To assess the feasibility of sentinel lymph node (SLN) localization and to determine the predictive value of SLN biopsy for occult neck metastases in patients previously treated with surgery or radiation therapy and with N0 squamous cell carcinoma of the oral cavity or oropharynx. DESIGN: Prospective case series. SETTING: Tertiary academic hospital. PATIENTS: Eleven patients with T1 to T4 N0 squamous cell carcinoma of the oral cavity or oropharynx. INTERVENTIONS: Patients underwent preoperative peritumoral injection of technetium Tc 99m sulfur colloid followed by dynamic lymphoscintigraphy and operative localization of the SLN(s) with the use of a handheld gamma probe. MAIN OUTCOME MEASURES: The presence or absence of metastatic disease in N0 squamous cell carcinoma of the oral cavity and oropharynx in patients previously treated with surgery or radiation therapy as identified by SLN biopsy findings. RESULTS: In each of the 11 patients, 1 to 3 SLNs were identified by lymphoscintigraphy. All SLNs identified by lymphoscintigraphy were successfully identified and removed with the use of an intraoperative gamma probe. In 10 of the 11 patients, the biopsy findings from the SLN(s) accurately predicted the presence or absence of occult neck metastasis. There was 1 instance of a negative SLN with a positive neck dissection. The overall negative predictive value of the study was 91%. No aberrant lymphatic drainage patterns were observed in this study. CONCLUSION: In patients previously treated with surgery or radiation therapy, SLN biopsy was as effective as in previously untreated patients according to published reports and warrants inclusion of this patient group into larger studies.

Sentinel lymph node biopsy in N0 squamous cell carcinoma of the oral cavity and oropharynx.

OBJECTIVES: To ascertain the feasibility of sentinel lymph node (SLN) localization by preoperative lymphoscintigraphy and intraoperative gamma probe radiolocalization and to determine the predictive value of the SLN for occult metastasis of the neck in N0 squamous cell carcinoma of the oral cavity and oropharynx. DESIGN: A prospective study of 20 consecutive patients with N0 squamous cell carcinoma of the head and neck who underwent lymphoscintigraphy and SLN biopsy. INTERVENTIONS: On the day before surgery, each patient who completed the study underwent a submucosal peritumoral injection of unfiltered technetium 99m sulfur colloid followed by lymphoscintigraphy. Focal areas of radioactivity were marked on the overlying skin. The following day, the patients underwent resection of the primary tumor, elevation of subplatysmal flaps, identification and removal of the SLNs as identified by gamma probe, and complete neck dissections. RESULTS: Lymphoscintigraphy and gamma probe radiolocalization accurately identified 1 or more SLNs in all 20 patients. In 4 (20%) of the 20 patients, the SLN correctly identified metastatic disease. In no instance was the SLN negative when the lymphadenectomy specimen was positive. CONCLUSIONS: In this study, the SLN had a negative predictive value of 100%. Sentinel lymph node biopsy is feasible and appears to accurately predict the presence of occult metastatic disease. Although further study is warranted, SLN biopsy could potentially guide head and neck oncologists to the patient with N0 disease who would benefit most from selective neck dissection and prevent the morbidity of unnecessary neck dissection.